Host serum amyloid A1 facilitates Streptococcus pneumoniae adaptation to acidic stress induced by pneumococcal anaerobic metabolism

Host serum amyloid A1 facilitates Streptococcus pneumoniae adaptation to acidic stress induced by pneumococcal anaerobic metabolism | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Host serum amyloid A1 facilitates Streptococcus pneumoniae adaptation to acidic stress induced by pneumococcal anaerobic metabolism Weichen Gong, Masayuki Ono, Tomoko Sumitomo, Momoko Kobayashi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5862113/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Serum amyloid A1 (SAA1), an acute-phase protein, exhibits a decreased level in bronchoalveolar lavage fluid (BALF) following nasal Streptococcus pneumoniae infections. However, the function of SAA1 in relation to S. pneumoniae remains to be unclear. In this study, we investigate whether S. pneumoniae utilized SAA1 for its survival in host or not. We explored the function of SAA1 in relation to S. pneumoniae by initially comparing serum with high SAA1 levels to serum with low SAA1 levels. We then validated our findings using recombinant SAA1. In this research, we developed an original PBS-based intranasal administration method to induce SAA1 increases in serum. This innovative approach provides a convenient platform for studying the role of SAA1 in various bacterial species. In vitro experiments showed that when serum-THY broth was incubated with S. pneumoniae , a similar reduction in SAA1 was observed, which was proved to be independent of protease activity. Intake of AF488 (green-fluorescent probe)-conjugated recombinant SAA1 by S. pneumoniae was observed using fluoresence microscope. Then, we proved that intake of SAA1 enhanced S. pneumoniae resistance against formate, a key metabolite of its anaerobic metabolism. This study is the first to reveal the relationship between SAA1 and a Gram-positive bacterium. We demonstrate that S. pneumoniae exploits host acute-phase proteins to enhance its survival in formic acidic environments. These findings expand our understanding of bacterial survival strategies, highlighting how pathogens utilize host-derived components to adapt to hostile conditions. Biological sciences/Microbiology/Bacteria/Bacterial host response Biological sciences/Microbiology/Bacteria/Bacterial physiology Biological sciences/Microbiology/Bacteria/Bacterial synthetic biology Serum amyloid A1 Streptococcus pneumoniae formic acid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Streptococcus pneumoniae is the most common pathogen responsible for community-acquired pneumonia, bacteremia, and sepsis. 1 , 2 Upon infection, S. pneumoniae triggers a complex immune response characterized by the activation of both innate and adaptive immunity. This pathogen first encounters physical barriers and innate immune cells, such as alveolar macrophages, neutrophils, and dendritic cells, in the respiratory tract. Upon recognition by pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), these cells initiate a cascade of inflammatory signaling pathways. 3 Neutrophils are the earliest and most abundantly recruited leukocytes in S. pneumoniae infection. 4 The dysfunction of neutrophils leads to the higher susceptibility of mice infected with S. pneumonia e, indicating their importance in defending S. pneumoniae infection. 5 Additionally, neutrophils also mediate phagocytosis and bacterial killing through reactive oxygen species (ROS) production and the release of antimicrobial peptides. 6 Although neutrophil plays a crucial role in S. pneumoniae acute infection phase, the overactivation of neutrophils cause the extravasate accumulation in alveoli and excessive neutrophils even cause edematous and hemorrhagic lungs, which is a symptom in some bacterial pneumoniae patients. 7 The acute pulmonary edema reduces normal oxygen movements through the lungs, thus alveoli turn to be anaerobic environments. 8 As a facultative anaerobic organism, S. pneumoniae lacks the cytochromes and heme containing proteins involved in aerobic respiration. Although S. pneumoniae lack mechanisms for coping with oxidative stress, it is aerotolerant and showed capacity to survive under atmospheric oxygen pressure. 9 The first step of pneumococcal aerobic pathway is to convert pyruvate to acetyl phosphate, which catalyzed by pyruvate oxidase (SpxB). SpxB catalyzes a two-electron reduction of O 2 , thereby forming the potentially damaging compound H 2 O 2 . 10 In addition, S. pneumoniae is known to maintain a fermentative metabolism in anaerobic environments, it converts pyruvate to lactate via the lactate dehydrogenase (LDH). 11 Besides lactate, most of the pyruvate can be converted to acetyl-CoA and formate by the oxygen-sensitive pyruvate formate lyase (PFL). 12 Despite strong pneumococcal survival capacity in acidic stress conditions, such as the acidic environment of inflammatory foci, and tolerates lethal pH through a mechanism known as the acid tolerance response. S. pneumoniae activated acidic-stress induced lysis in response to acidic environments, favoring the release of cell wall compounds, DNA and virulence factors. 13 Besides immunocytes, acute-phase proteins (APPs) are also rapidly synthesized by the liver in response to S. pneuomoniae infection. These APPs are often triggered by pro-inflammatory cytokines like IL-6 and TNF-α. 14 Serum amyloid A1 (SAA1) is one such acute-phase protein, and its role during pneumococcal infections has recently garnered attention. SAA1 is known for its involvement in lipid metabolism and inflammatory responses, but its exact role in modulating the immune response during bacterial infections remains unclear, especially in Gram-positive bacterium. 15 In previous studies, SAA1 was reported to bind to outer membrane protein A (OmpA) of Gram-negative bacteria, and promotes LPS clearance as well as suppresses LPS-induced inflammation and tissue injury. 16 , 17 Given the fact that SAA1 participate in Gram-negative bacterial infection, we have interest in investigating how SAA1 works during S. pneumoniae , a Gram-positive bacterial infection. In this study, we focus on elucidating the relationship between S. pneumoniae infection and SAA1, and found that S. pneumoniae enhance its resistance againsit formatic acidic stress by utilizing host SAA1. Results Decreased levels of SAA1 in BALF, not in serum following S. pneumoniae challenge in mice. Pneumococcal infection progresses through three distinct stages, each associated with varying symptoms. First, S. pneumoniae resides in the nasopharynx and patients are typically asymptomatical, Next, if S. pneumoniae migrate to the lungs and patient presented with symptoms of pneumonia, leading to acute pulmonary edema. Eventually, if S. pneumoniae breaches the pulmonary epithelium and enters the bloodstream, patients The patient will be diagnosed with sepsis in some severe cases. 8 To investigate the host response to pneumococcal infection, we challenged mice intranasally with the S. pneumoniae D39 strain (virulent serotype 2) and collected bronchoalveolar lavage fluid (BALF) and blood 24 hours post-infection (Fig. 1 A). In this intranasal infection model, we observed a significant decrease in SAA1 levels in BALF, while C-reactive protein (CRP) levels remained stable (Fig. 1 B, C). Consequently, we focused on SAA1 for subsequent experiments. Despite the reduction of SAA1 in BALF, no changes in serum SAA1 levels were detected in intranasally infected mice (Fig. 1 D). This is likely because S. pneumoniae did not breach the pulmonary barrier, as no bacteria were detected in the blood 24 hours post-infection (Fig. 1 E). To examine the effects of S. pneumoniae on SAA1 levels in the bloodstream, we used an intravenous infection model. Mice were challenged with the S. pneumoniae D39 strain, and bacterial loads in the spleen, liver, lungs, and blood were quantified (Fig. 2 A). S. pneumoniae was successfully detected in the blood, and bacterial distribution across the spleen, liver, and lungs showed no significant differences 24 hours post-infection, confirming the successful establishment of the sepsis model (Fig. 2 B; Fig. S1 ). However, SAA1 levels in the serum remained unchanged between infection and non-infection groups (Fig. 2 C). These results demonstrate that intranasal infection with S. pneumoniae reduces SAA1 levels in BALF without affecting serum SAA1, likely due to the bacteria's inability to enter the bloodstream. In contrast, intravenous infection did not alter serum SAA1 levels, and the underlying reason requires further investigation in future studies. S. pneumoniae reduces SAA1 levels via intake rather than catalysis. To investigate how S. pneumoniae causes the reduction of SAA1 in BALF, we utilized non-infected mouse serum diluted with THY broth medium for in vitro experiments, as serum contains abundant SAA1 compared to BALF. Initially, we tested whether this phenomenon is observed across various S. pneumoniae strains. Serum-THY broth (10% serum, v/v) was co-cultured with two commonly used S. pneumoniae strains, D39 (virulent serotype 2) and TIGR4 (virulent serotype 4). Since SAA1 is an amyloid protein with a β-sheet structure prone to aggregation, we added formic acid (0.05%, v/v), which prevents aggregation of SAA1 under conditions that bacteria remains growing (Fig. S2). Results showed that the TIGR4 strain had a stronger capacity to reduce SAA1 compared to the D39 strain (Fig. 3 A). Consequently, TIGR4 was selected for further investigation in studying SAA1 reduction. By measuring SAA1 levels in bacterial culture medium using ELISA, we found that formic acid can promote the SAA1 reduction by S. pneumoniae . This result indicates that formic acid (0.05%, v/v) was crucial for studying SAA1 reduction with the TIGR4 strain, as no reduction occurred in its absence (Fig. 3 B). Previous studies, such as those by Zhiming Wang et al., reported that S. pneumoniae secretes a giant metalloprotease (IgA1 protease) to cleave host IgA1. 19 We hypothesized that a pneumococcal protease might mediate SAA1 reduction. However, the addition of protease inhibitor to the culture system failed to prevent SAA1 reduction, suggesting that this process is independent of pneumococcal proteases (Fig. 3 C). We next hypothesized that SAA1 might bind to the S. pneumoniae cell membrane, a mechanism previously reported in Gram-negative bacteria. To localize SAA1, we constructed AF488-conjugated recombinant mouse SAA1 (rmSAA1) and incubated it with TIGR4. Fluorescence microscopy revealed that AF488-conjugated rmSAA1 bound to the S. pneumoniae cell membrane after 2 hours of incubation, and parts of it were internalized by S. pneumoniae after 3 hours (Fig. 3 D). Additionally, enhanced fluorescence intensity were observed after permeabilization of S. pneumoniae co-cultured with AF488-rmSAA1 for 3 hours (Fig. S3). These observations indicate that SAA1 reduction is due to the intake by S. pneumoniae , rather than being cleaved by bacterial proteases. These results suggest that the reduction of SAA1 by S. pneumoniae existing in both D39 and TIGR4 strains, but TIGR4 showing a stronger effect than D39. The process is possibly facilitated by formic acid and is independent of pneumococcal proteases. Instead, SAA1 reduction involves its binding to and subsequent internalization, and consequently intake by S. pneumoniae . SAA1 uptake improves S. pneumoniae resistance to formic acidic environment. As a 122-amino-acid protein, SAA1 is internalized by S. pneumoniae and potentially utilized as a source of amino acids. In previous studies, commensal and pathogenic E. coli were shown to overcome extreme acidic environments (pH < 2.5) through a glutamate-dependent mechanism involving glutamate decarboxylase (GadB), which catalyzes a proton-consuming decarboxylation reaction, and the antiporter GadC, which facilitates glutamate in /γ-aminobutyrate (GABA) out exchange. This glutamate-dependent acid resistance (GDAR) system is also present in Shigella flexneri , Listeria monocytogenes , and Lactococcus lactis . 20 Based on this, we hypothesized that SAA1 intake might improve S. pneumoniae resistance to acidic stress in anaerobic environments. S. pneumoniae , like other pathogenic bacteria, must adapt to diverse and often hostile environments within the host. During acute infections, inflammatory responses lead to pulmonary edema, which impairs oxygen exchange and creates anaerobic conditions in the alveoli. 8 We observed that culture medium became more acidic when S. pneumoniae D39 was cultured anaerobically compared to aerobically (Fig. 4 A). Previous studies reported increased production of acetate, lactate, and formate by S. pneumoniae under anaerobic conditions (Fig. 4 B). We tested the resistance of S. pneumoniae D39 to these acids and determined the maximum non-inhibitory concentrations, that are 0.05% (v/v) formic acid, 0.125% (v/v) lactic acid, and 0.083% (v/v) acetic acid (Table S1 -3). We collected serum and measured SAA1 levels using ELISA, then seperated serum into serum high−SAA1 (400 ~ 1000 µg/mL SAA1) and serum low−SAA1 (10 ~ 20 µg/mL SAA1) groups. To investigate whether SAA1 facilitates S. pneumoniae resistance to acidic stress, we cultured S. pneumoniae D39 in lethal concentrations of formic (0.075%, v/v), lactic (0.1875%, v/v), and acetic (0.125%, v/v) acids with 10% serum containing either serum high−SAA1 or serum low−SAA1 . Both serum high−SAA1 and serum low−SAA1 enhanced S. pneumoniae D39 resistance to lethal formic acid (0.075%, v/v), with serum high−SAA1 promoting better bacterial growth after 10 hours compared to serum low−SAA1 (Fig. 4 C). However, SAA1 contributed minimally to resistance against acetic and lactic acids (Fig. S4A, S4B). To further confirm that SAA1 uptake enhances resistance to formic acid, we cultured S. pneumoniae with AF488-rmSAA1 and used propidium iodide to stain dead cells. Propidium iodide, which penetrates the compromised membranes of dead bacteria, binds to DNA and fluoresces. 21 Under fluorescence microscopy, few green bacteria (indicating AF488-rmSAA1 intake) were stained with propidium iodide after 2 hours of exposure to lethal formic acid (0.25%,v/v) (Fig. 4 D; Fig. S5). This result aligns with the growth curve findings (Fig. 4 C, E), leading to the conclusion that SAA1 intake improves S. pneumoniae resistance to formic acid stress. It should be noted that serum low−SAA1 was obtained from untreated 8-week-old C57BL/6 mice, while serum high−SAA1 was derived from mice treated with intranasal PBS administration, which significantly elevated serum SAA1 levels (Fig. 5 A, B, C). It is an original PBS-based intranasal administration method to induce SAA1 increases in serum. This innovative approach provides a convenient platform for studying the role of SAA1 in various bacterial species. These results indiciate that SAA1 intake by S. pneumoniae enhances bacterial resistance to formic acid stress in anaerobic environments. High SAA1 concentrations in serum significantly boost this resistance, while the effect on resistance to acetic and lactic acids is minimal (Fig. 6 ). Discussion During S. pneumoniae infection, the bacterium triggers a complex immune response involving both innate and adaptive immunity. Neutrophils, as the earliest and most abundant leukocytes recruited to the site of infection, play a critical role. Meanwhile, dendritic cells process bacterial antigens and present them to T cells, initiating an adaptive immune response. Among these, T helper 17 (Th17) cells, a subset of CD4 + T cells, are pivotal in producing IL-17, which promotes neutrophil recruitment and enhances mucosal defense against S. pneumoniae . Additionally, B cells generate specific antibodies that facilitate opsonization, neutralize the bacterial capsule, and promote pathogen clearance through complement activation. The development of memory B cells and durable antibodies is particularly important for preventing reinfection and improving vaccine efficacy. 22 , 23 While the roles of various immune cells in S. pneumoniae infection have been extensively studied, there is limited understanding of how acute-phase proteins (APPs) function alone or in coordination with immune cells during such infections. C-reactive protein (CRP), for instance, is known to bind phosphocholine on the bacterial surface, promoting opsonization and complement-mediated clearance. 24 Although widely studied as a biomarker of bacterial infection and inflammation, CRP's specific role in pneumococcal immunity highlights the importance of systemic acute-phase responses. To date, the function of SAA1 has been explored mainly in Gram-negative bacterial infections. For example, SAA1 has been reported to bind to outer membrane protein A (OmpA) of Gram-negative bacteria, promoting lipopolysaccharide (LPS) clearance and suppressing LPS-induced inflammation and tissue injury. 16 , 17 In this study, we investigated the role of SAA1 in S. pneumoniae infection. We observed that nasal infection with S. pneumoniae led to a reduction of SAA1 in mice BALF (Fig. 1 c). Similarly, when S. pneumoniae D39 and TIGR4 strains were co-cultured with serum-THY broth (10% serum, v/v) for 12 hours in vitro , SAA1 levels decreased, with the TIGR4 strain showing a stronger capacity to reduce SAA1 (Fig. 3 a). Consequently, we selected the TIGR4 strain as a model to study the mechanism underlying this reduction. Since S. pneumoniae produces IgA1 protease, we hypothesized the presence of an SAA1-specific protease. However, adding protease inhibitors to the culture system failed to prevent SAA1 reduction (Fig. 3 c). Instead, we observed that AF488-rmSAA1 started to be internalized by S. pneumoniae after three hours of co-culture (Fig. 3 d). These findings suggest that S. pneumoniae reduces SAA1 levels in BALF by actively intaking SAA1. Interestingly, we found that formic acid is necessary for SAA1 intake by S. pneumoniae TIGR4, though the underlying mechanism remains unclear. Initially, we added formic acid (0.05%,v/v) to the culture system to prevent SAA1 aggregation. The results suggest that formic acid may activate S. pneumoniae to internalize SAA1, potentially aiding its adaptation to formic acid stress. However, no receptor or protein capable of sensing formic acid concentrations has been identified in either prokaryotes or eukaryotes. Formic acid is commonly used as a preservative and antibacterial agent in livestock feed, and it is also produced as a short-chain fatty acid (SCFA) by intestinal microorganisms during metabolism. 25 Although acetate, propionate, and butyrate constitute the majority of SCFAs in the human colon, formic acid is present in smaller amounts and may be associated with methanogenesis or elevated during inflammation. Its physiological and pathological roles remain largely unexplored, highlighting the need for further investigation. 26 , 27 One challenge in studying SAA1 function is the high cost of recombinant SAA1 due to the difficulties in purifying it from E. coli expression systems, stemming from its fibrillation and amphiphilic properties. 28 Previous methods for inducing SAA1 production in mice, such as subcutaneous silver nitrate injections or intranasal LPS administration, are either hazardous or costly and can interfere with experimental outcomes by inducing antibody production. 29 , 30 In this study, we developed a cost-effective and stable method to prepare SAA1-abundant serum by intranasal administration of PBS in male C57BL/6 mice. This approach successfully induced SAA1 production, although the underlying mechanism remains unclear. We collected serum with high SAA1 levels (serum high−SAA1 ) from PBS-treated mice and serum with low SAA1 levels (serum low−SAA1 ) from untreated mice. This method enabled us to compare the effects of SAA1 using serum samples first and then confirm the results with recombinant SAA1. The glutamate-dependent acid resistance (GDAR) system, found in Shigella flexneri , Listeria monocytogenes , and Lactococcus lactis , facilitates bacterial adaptation to acidic stress. 20 Here, we investigated whether SAA1 could enhance S. pneumoniae resistance to acidic stress. During pneumonia, S. pneumoniae transitions from the nasopharynx to the lungs, where fluid accumulation and oxygen reduction create anaerobic conditions. Previous studies have shown that S. pneumoniae produces acetate, lactate, and formate under anaerobic conditions (Fig. 4 b). We found that the pH of S. pneumoniae cultures was lower under anaerobic conditions than aerobic ones (Fig. 4 a). Due to technical limitations, we could not identify specific metabolites but selected acetate, lactate, and formate as representative byproducts. Using serum high−SAA1 and serum low−SAA1 , we observed that S. pneumoniae D39 displayed better growth in lethal concentrations of formic acid (0.075%, v/v) when co-cultured with serum high−SAA1 compared to serum low−SAA1 (Fig. 4 c, e). This finding was further confirmed using AF488-rmSAA1 and fluorescence microscopy, which demonstrated that SAA1 intake enhances S. pneumoniae resistance to formic acid stress (Fig. 4 d). In conclusion, our study reveals a novel adaptation strategy employed by S. pneumoniae to cope with formic acid stress by internalizing host SAA1 (Fig. 6 ). This interaction between an acute-phase protein and a bacterial pathogen enhances the pathogen’s ability to resist acidic conditions. By utilizing SAA1, an acute-phase protein associated with immune defense and inflammation, S. pneumoniae gains a survival advantage in acidic environments, improving its fitness during infection and evading metabolic stress. This discovery expands our understanding of pneumococcal survival strategies and highlights the importance of host-pathogen interactions in shaping bacterial adaptation. Methods Our research complied with all of the relevant ethical regulations. All of the animal procedures were conducted according to the protocols approved by Animal Care and Use Committee of Osaka University Graduate School of Dentistry (authorization number 04-018-0). All methods are reported in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) to ensure rigorous and transparent reporting of animal research ( https://arriveguidelines.org ). Bacterial culture Streptococcus pneumoniae D39 (serotype 2 clinical isolate) and TIGR4 (virulent serotype 4 clinical isolate) strains were cultured in Todd-Hewitt broth (Becton, Dickinson and Company [BD], New Jersey, U.S.) supplemented with 0.2% yeast extract (BD) (THY broth) at 37°C in 5% CO 2 incubator. Animal infection experiments Female Slc:ICR mice at 6 to 7 weeks old (SLC Japan, Inc., Shizuoka, Japan) were used as animal infection model in this study. The S. pneumoniae D39 strain was grown to the early-exponential phase (OD 600 of 0.1) and then washed with and resuspended in PBS. For intranasal infection experiments, each mouse was intraperitoneally given 250 µL of anaesthetic consisting of 20 µL midazolam (Takeda Pharmaceuticals, Osaka, Japan), 7.5 µL domitor (ZENOAQ, Fukushima, Japan), 25 µL vetorphale (Meiji Animal Health Co., Ltd., Kumamoto, Japan) and 197.5 µL PBS solution, then mice were given to mice by administration of 4 × 10 6 CFU in 20 µL PBS. Blood and BALF were collected from mice 24 hours post intranasal infection after mice were euthanized by intraperitoneal injection of 300 µL pentobarbital sodium solution (20 mg/mL, Product mumber: P0776, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan). BALF were collected by slowly drawing the injected PBS back into the syringe and the serum were collected from blood by centrifugation (8,500 rpm, 15 minutes, 4 ˚C). In intravenous infection experiments, bacteria were introduced into mice by administration of 2 × 10 5 CFU in 100 µL PBS. For quantification of bacterial colonization in the lung, liver, spleen and blood, mice were euthanized 24 hours after S. pneumoniae infection by intraperitoneal injection of 300 µL pentobarbital sodium solution (20 mg/mL, Product mumber: P0776, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) and then lung, liver, spleen tissues and blood were immediately collected. Tissue homogenates and blood were serially diluted and plated on THY agar plates and then incubated at 5% CO 2 incubator. After 24 hours incubation, colony-forming unit (CFU) per weight of organ tissues (gram) or volume of blood (mL) was calculated. Quantification of SAA1 and C-reaction protein (CRP) using ELISA After the collection of serum and BALF from mice, SAA1/2 and CRP quantification was performed by ELISA following the manufacturer's protocols described in mouse serum amyloid A ELISA Kit (SAA; Cat# KMA0021, Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, U. S.) and mouse CRP ELISA Kit (Cat# EM20RB, Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, U. S.). In SAA1 reduction in vitro experiments (Fig. 3 B, C), we utilized absorbance intensity (OD 450 ) instead of quantification to determine SAA1 levels. Preparation of serum high−SAA1 and serum low SAA 1 Male C57BL/6J mice at 7 to 8 weeks old (SLC Japan, Inc., Shizuoka, Japan) were used as source of serum high−SAA1 and serum low−SAA1 . 20 µL of 1x phosphate-buffered saline (PBS) were given to mice intranasally. After 24 hours, mice were euthanized by intraperitoneal injection of 300 µL pentobarbital sodium solution (20 mg/mL, Product mumber: P0776, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), and cardiac puncture was performed to collect blood. The serum was collected from blood by centrifugation (8,500 rpm, 15 minutes, 4 ˚C). SAA1 concentration of each sample were determined by mouse serum amyloid A ELISA Kit (SAA; Cat# KMA0021, Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, U. S.). This method was confirmed to significantly and stably increase the serum SAA1 level. Thus, serum high−SAA1 were collected from PBS-intranasal administration and serum low−SAA1 were collected from mice without PBS-intranasal administration. Co-culture of S. pneumoniae with SAA1-abundant serum in vitro The SAA1-abundant serum from PBS-intranasal administration C57BL/6J mice and the SAA1 concentration were measured by mouse serum amyloid A ELISA Kit (SAA; Cat# KMA0021, Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, U. S.). S. pneumoniae D39 strain or TIGR4 strain was grown to the early-exponential phase (OD 600 of 0.1). Then, 5 µL of bacterial culture were added into 20 µL serum and 175 µL THY broth (containing final 0.05% formic acid, v/v) in a 96-well plate and incubated in 5% CO 2 incubator for 12 hours. To test whether SAA1 reduction was protease-dependent, protease inhibitor (cOmplete™, EDTA-free Protease Inhibitor Cocktail, Roche, Swiss) was dissolved and used following its instruction for use. After incubation, the supernatent of bacterial culture were collected after centrifugation (12,000 rpm, 10 minutes, 4 ˚C) and the remaining SAA were measured using mouse serum amyloid A ELISA Kit (SAA; Cat# KMA0021, Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, U. S.). AF488-conjugated recombinant SAA1 intake by S. pneumoniae observation using fluorescence microscope Recombinant mouse SAA1(rmSAA1) (Cat# 2948-SA-025, R&D Systems, Minneapolis, U.S.) were conjugated with AF488 following the protocol described in Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (Abcam, Cat# ab236553, Abcam Limited, Cambridge, U. K.). S. pneumoniae TIGR4 strain was grown to the early-exponential phase (OD 600 of 0.1) and 1 mL of bacteria were collected and resuspended with 50 µL PBS containing AF488-rmSAA1 (final concentration = 0.25 µg/mL) with existence 0.05% formic acid (concentration that not inhibiting pneumococcal growth) or 0.25% formic acid (lethal concentration). Then, bacteria were incubated in 5% CO 2 incubator, 5 µL of bacteria were collected after 1 hour, 2 hours and 3 hours, then were observed using fluorescence microscope (KEYENCE BZ-X, Osaka, Japan). In Fig. S3, we collected the bacterial pellet at 3 hours post incubation through centrifugation (12,000 rpm, 10 minutes, 4 ˚C) and then added 0.05% Triton X-100 into bacterial pellet to permeabilize bacteria, then its fluorescence intensity (Ex/Em = 485/535) was measured after permeabilization for 0, 0.5, 1 hour. In Fig. S4, we cultured S. pneumoniae in 0.25% formic acid together with AF488-rmSAA1 (final concentration = 0.25 µg/mL). After 2 hours incubation, Propidium Iodide was added to bacteria (final concentration = 1 µg/mL) and incubated at 37 ˚C for 15 minutes. After incubation, bacteria were washed twice and resuspended with PBS. Then, the bacteria were observed under fluorescence microscope (KEYENCE BZ-X, Osaka, Japan). pH measurement of bacterial culture under aerobic and anaerobic environment Streptococcus pneumoniae D39 were added in a 24-well plate, then cultured in aerobic (5% CO 2 incubator) and anaerobic environment created by using AnaeroPack-Anaero (MITSUBISHI GAS CHEMICAL Co. Inc, Japan). The supernatant of bacterial cultures was collected at different time points (1, 3, 5, 7, 9 hours). 50 µL of 0.04% (w/v) phenol red broth (Cat# 165–01121, FUJIFILM Wako Pure Chemical Corporation, Japan) were mixed with 950 µL bacterial supernatant collected at each point was measured the absorbance at 560 nm. A calibration curve was determined in phenol red broth which adjusted to pH values ranging from 4 to 10. For THY broth, culture supernatant at each point was supplemented with 0.04% phenol red and the absorbance was measured at 550 nm. A calibration curve was determined in THY broth which was supplemented with 5 mg/mL phenol red and adjusted to pH values ranging from 3 to 10. Bacterial growth curve in lethal formic, lactic and acetic acid concentration. S. pneumoniae D39 strain was grown to the early-exponential phase (OD 600 of 0.1). Then, 5 µL of bacterial culture were added into 20 µL serum high−SAA1 and serum low−SAA1 and 175 µL THY broth (containing 0.125% acetic, 0.1875% lactic, and 0.075% formic acid, v/v) in a 96-well plate and incubated in 5% CO 2 incubator for 12 hours. The OD 600 of each well was recorded by microplate reader (Infinite® M Plex, Tecan, Swiss). Statistical analysis All results were statistically analysed using the Student's t- test in Excel (v. 16.81; Microsoft, Redmond, WA, USA). Differences were considered statistically significant at p < 0.05. Data visualization Hiplot (ORG) ( https://hiplot.org ), a comprehensive and easy-to-use web service for boosting publication-ready biomedical data visualization, was used to visualize the following data: Fig. 1 B-E; Fig. 2 B, C; Fig. 3 A, C; Fig. 5 B, C; Fig. S1 . The other data were created by Excel (v. 16.81; Microsoft, Redmond, WA, USA) and modified by Keynote (v. 14.1; Apple Inc., California, U. S.) Declarations Acknowledgements We thank the OpenBiox community and the Hiplot team (https://hiplot.org) for providing technical assistance and valuable tools for data analysis and visualization. Funding This study was supported by JST SPRING (grant number JPMJSP2138). The funders had no role in the study design, data collection or analysis, decision to publish, or preparation of the manuscript. Author contributions W.G. and S.K. designed the study. W.G., M.O., and K.M. performed the experiments. T.S. and Y.H. gave their valuable and helpful suggestion. W.G. wrote the original manuscript. All authors reviewed and edited the manuscript. Data availability statement The datasets generated in this study are available from the corresponding author upon request. Declaration of Competing Interest The authors have no competing interests to declare. Ethical approval statement All mouse experiments were conducted using a protocol approved by the Animal Care and Use Committee of Osaka University Graduate School of Dentistry (authorization number 04-018-0). References Austrian, R. Some aspects of the pneumococcal carrier state. J. Antimicrob. Chemother. 18 (Suppl A), 35–45 (1986). Musher, D. M. How contagious are common respiratory tract infections? N Engl. J. Med. 348 , 1256–1266 (2003). Koppe, U., Suttorp, N. & Opitz, B. Recognition of Streptococcus pneumoniae by the innate immune system. Cell. Microbiol. 14 (4), 460–466 (2012). Craig, A. et al. Neutrophil recruitment to the lungs during bacterial pneumonia. Infect. 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Effect of hydrogen peroxide production and the Fenton reaction on membrane composition of Streptococcus pneumoniae. Biochim. Biophys. Acta . 1768 (3), 590–597 (2007). Neijssel, O. M. & Snoep, J. L. M. J. Teixeira de Mattos. Regulation of energy source metabolism in streptococci. Soc. Appl. Bacteriol. Symp. Ser. 26, 12S-19S (1997). Yesilkaya, H. et al. Pyruvate formate lyase is required for pneumococcal fermentative metabolism and virulence. Infect. Immun. 77 (12), 5418–5427 (2009). Cortes, P. R. et al. Stress-triggered signaling affecting survival or suicide of Streptococcus pneumoniae. Int. J. Med. Microbiol. 305 (1), 157–169 (2015). Moshage, H. Cytokines and the hepatic acute phase response. J. Pathol. 181 (3), 257–266 (1997). Zhang, Y. et al. Acute phase reactant serum amyloid A in inflammation and other diseases. Adv. Clin. Chem. 90 , 25–80 (2019). Hari-Dass, R. et al. Serum amyloid A protein binds to outer membrane protein A of gram-negative bacteria. J. Biol. Chem. 280 (19), 18562–18567 (2005). Cheng, N. et al. Serum amyloid A promotes LPS clearance and suppresses LPS-induced inflammation and tissue injury. EMBO Rep. 19 (10), e45517 (2018). Christensen, L. F. B. et al. Quantitating denaturation by formic acid: Imperfect repeats are essential to the stability of the functional amyloid protein FapC. J. Biol. Chem. 295 (37), 13031–13046 (2020). Wang, Z. et al. Mechanism and inhibition of Streptococcus pneumoniae IgA1 protease. Nat. Commun. 11 (1), 6063 (2020). De Biase, D. & Pennacchietti, E. Glutamate decarboxylase-dependent acid resistance in orally acquired bacteria: function, distribution and biomedical implications of the gadBC operon. Mol. Microbiol. 86 (4), 770–786 (2012). Riccardi, C. & Nicoletti, I. Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat. Protoc. 1 (3), 1458–1461 (2006). Bogaert, D., De Groot, R. & Hermans, P. W. Streptococcus pneumoniae colonisation: the key to pneumococcal disease. Lancet Infect. Dis. 4 (3), 144–154 (2004). Ramos-Sevillano, E., Ercoli, G. & Brown, J. S. Mechanisms of naturally acquired immunity to Streptococcus pneumoniae. Front. Immunol. 10 , 358 (2019). Olson, M. E. et al. A biofunctional review of C-reactive protein (CRP) as a mediator of inflammatory and immune responses: differentiating pentameric and modified CRP isoform effects. Front. Immunol. 14 , 1264383 (2023). Ríos-Covián, D. et al. Intestinal short chain fatty acids and their link with diet and human health. Front. Microbiol. 7 , 185 (2016). Bereswill, S. et al. Novel murine infection models provide deep insights into the menage a trois of Campylobacter jejuni, microbiota and host innate immunity. PloS one , 6 (6), e20953 (2011). Vanderhaeghen, S., Lacroix, C. & Schwab, C. Methanogen communities in stools of humans of different age and health status and co-occurrence with bacteria. FEMS Microbiol. Lett. 362 (13), fnv092 (2015). Ahmad, S. et al. Nature of recombinant human serum amyloid A1 in Escherichia coli and its preferable approach for purification. Protein Expr Purif . Published online November 5 (2024). Brissette, L. et al. Differential induction of the serum amyloid A gene family in response to an inflammatory agent and to amyloid-enhancing factor. J. Biol. Chem. 264 (32), 19327–19332 (1989). Wang, S. et al. Establishment of a mouse model of lipopolysaccharide-induced neutrophilic nasal polyps. Exp. Ther. Med. 14 (6), 5275–5282 (2017). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5862113","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":418491173,"identity":"6ef58602-f5fc-46be-9280-f8d735e2fc05","order_by":0,"name":"Weichen Gong","email":"data:image/png;base64,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","orcid":"","institution":"Osaka University Graduate School of Dentistry","correspondingAuthor":true,"prefix":"","firstName":"Weichen","middleName":"","lastName":"Gong","suffix":""},{"id":418491174,"identity":"93a9d007-fd74-4a82-9908-18f8086bd33a","order_by":1,"name":"Masayuki Ono","email":"","orcid":"","institution":"Osaka University Graduate School of Dentistry","correspondingAuthor":false,"prefix":"","firstName":"Masayuki","middleName":"","lastName":"Ono","suffix":""},{"id":418491176,"identity":"01cff3a8-7de9-488c-a31c-2f31c1794129","order_by":2,"name":"Tomoko Sumitomo","email":"","orcid":"","institution":"Tokushima University","correspondingAuthor":false,"prefix":"","firstName":"Tomoko","middleName":"","lastName":"Sumitomo","suffix":""},{"id":418491180,"identity":"72bd8829-f36f-4fb5-94e8-99d673dff39d","order_by":3,"name":"Momoko Kobayashi","email":"","orcid":"","institution":"Osaka University Graduate School of Dentistry","correspondingAuthor":false,"prefix":"","firstName":"Momoko","middleName":"","lastName":"Kobayashi","suffix":""},{"id":418491182,"identity":"75921c3f-2b01-436d-952c-ca323fee425f","order_by":4,"name":"Yujiro Hirose","email":"","orcid":"","institution":"Osaka University Graduate School of Dentistry","correspondingAuthor":false,"prefix":"","firstName":"Yujiro","middleName":"","lastName":"Hirose","suffix":""},{"id":418491183,"identity":"295f7f51-1966-4b4e-880b-5d10f5dc2c72","order_by":5,"name":"Shigetada Kawabata","email":"","orcid":"","institution":"Osaka University Graduate School of Dentistry","correspondingAuthor":false,"prefix":"","firstName":"Shigetada","middleName":"","lastName":"Kawabata","suffix":""}],"badges":[],"createdAt":"2025-01-20 03:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5862113/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5862113/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76844650,"identity":"5330c427-0042-4bb9-af00-1fee8ac2f4e2","added_by":"auto","created_at":"2025-02-21 10:42:34","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":452532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSAA1 decreased but CRP remained stable in BALF collected from intranasal \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. pneumoniae \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003einfection mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The experimental workflow of intranasal \u003cem\u003eS. pneumoniae \u003c/em\u003einfection. (B) CRP in BALF displayed no significant difference between non-infection (PBS-intranasal administration, n=14) and infection (\u003cem\u003eS. pneumoniae \u003c/em\u003eD39-nasal administration, n=8) groups. ns, no significant difference; Student’s \u003cem\u003et\u003c/em\u003e-test used for statistical analysis. \u0026nbsp;(C) SAA1 in BALF decreased in infection (\u003cem\u003eS. pneumoniae \u003c/em\u003eD39-nasal administration, n=18) group in comparison to non-infection (PBS-intranasal administration, n=18) group. n=6; ns, no significant difference. *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; Student’s \u003cem\u003et\u003c/em\u003e-test used for statistical analysis. (D) SAA1 in serum displayed no significant difference between infection (\u003cem\u003eS. pneumoniae \u003c/em\u003eD39-nasal administration, n=6) and non-infection (PBS-intranasal administration, n=6) groups. ns, no significant difference; Student’s \u003cem\u003et\u003c/em\u003e-test used for statistical analysis. (E) Detection of \u003cem\u003eS. pneumoniae\u003c/em\u003e in both blood and BALF 24 hours post PBS- or \u003cem\u003eS. pneumoniae \u003c/em\u003eD39-nasal administration. n=18.\u003c/p\u003e","description":"","filename":"MainFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5862113/v1/91eb1b98fb176f4bb6f10ed2.jpg"},{"id":76846446,"identity":"38298824-81dd-46f9-8da7-cb3bf24cf199","added_by":"auto","created_at":"2025-02-21 10:58:34","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":330314,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSAA1 remained stable in serum collected from intravenous \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. pneumoniae \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003einfection mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The experimental workflow of intravenous \u003cem\u003eS. pneumoniae \u003c/em\u003einfection. (B) Detection of \u003cem\u003eS. pneumoniae\u003c/em\u003e in blood 24 hours post PBS- or \u003cem\u003eS. pneumoniae \u003c/em\u003eD39-nasal administration. n=18. (C) SAA1 in serum displayed no significant difference between infection (\u003cem\u003eS. pneumoniae \u003c/em\u003eD39-nasal administration, n=18) and non-infection (PBS-intranasal administration, n=18) groups. ns, no significant difference; Student’s \u003cem\u003et\u003c/em\u003e-test used for statistical analysis.\u003c/p\u003e","description":"","filename":"MainFigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5862113/v1/563f10709879630b1c0ab939.jpg"},{"id":76844652,"identity":"924da8aa-36b4-4409-8e2f-61481f23eb03","added_by":"auto","created_at":"2025-02-21 10:42:34","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":452131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eS. pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e reduces SAA1 levels via intake rather than catalysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) TIGR4 strain displayed stronger capacity to cause SAA1 reduction with existence of formic acid (0.05%, v/v) \u003cem\u003ein vitro, \u003c/em\u003ecompared to D39 strain. n=3; ns, no significant difference; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; Student’s \u003cem\u003et\u003c/em\u003e-test used for statistical analysis. (B) Formic acid (0.05%, v/v) is necessary for SAA1 reduction when SAA1-abundant serum coculturing with \u003cem\u003eS. pneumoniae\u003c/em\u003e TIGR4. n=3. (C) SAA1 reduction was not affected by the addition of protease inhibitor, which indicates SAA1 reduction was not mediated by bacterial protease. n=3. ns, no significant difference; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005; Student’s \u003cem\u003et\u003c/em\u003e-test used for statistical analysis. (D) Fluorescence microscope showed the location of AF488-recombinant mouse SAA1 (AF488-rmSAA1) at 1, 2, 3 hours post incubation.\u003c/p\u003e","description":"","filename":"MainFigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5862113/v1/19ff1bb776a75453076d34a2.jpg"},{"id":76845067,"identity":"77696d8b-a62c-4ba1-9330-bdaaeed0b714","added_by":"auto","created_at":"2025-02-21 10:50:34","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":468811,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntake of SAA1 facilitate \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. pneumoniae \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eadaption to formic acid, a pneumococcal anaerobic metabolite\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Bacterial cultures in anaerobic conditions showed more acidic in comparison to that in anerobic conditions. n=6. *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005; Student’s \u003cem\u003et\u003c/em\u003e-test used for statistical analysis. (B) Graphic summary of \u003cem\u003eS. pneumoniae \u003c/em\u003eanaerobic metabolism. (C) Growth curve of \u003cem\u003eS. pneumoniae \u003c/em\u003ein lethal concentration of formic acid with no serum, serum \u003csup\u003ehigh-SAA1\u003c/sup\u003e or serum \u003csup\u003elow-SAA1\u003c/sup\u003e. (D) Fluorescence microscope showed that intake of AF488-rmSAA1 keeps \u003cem\u003eS. pneumoniae\u003c/em\u003e alive in lethal concentration of formic acid. Green, AF488-rmSAA1; red, dead bacteria; orange, merge of red and green fluorescence. (E) CFU counting of \u003cem\u003eS. pneumoniae \u003c/em\u003eco-cultured with serum \u003csup\u003ehigh-SAA1\u003c/sup\u003e and serum \u003csup\u003elow-SAA1 \u003c/sup\u003eat 11 hours and 16 hours post incubation. n=4. ns, no significant difference; *, \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05; Student’s \u003cem\u003et\u003c/em\u003e-test used for statistical analysis.\u003c/p\u003e","description":"","filename":"MainFigure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5862113/v1/3c0cf74ecc1ba9fd18ad3e07.jpg"},{"id":76844654,"identity":"cc8dbc80-c5d2-4c85-b704-470c7f8a8e7d","added_by":"auto","created_at":"2025-02-21 10:42:34","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":444249,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePBS-intranasal administration increases the serum SAA1 concentration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The experimental workflow of PBS-intranasal administration to mice. (B) SAA1 concentration of pre- and post- intranasal administration of PBS or saline. Control group, n=7; PBS group, n=6; saline group, n=5. (C) Fold change of SAA1 level after intranasal administration of PBS or saline. Control group, n=7; PBS group, n=6; saline group, n=5. ns, no significant difference; *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; Student’s \u003cem\u003et\u003c/em\u003e-test used for statistical analysis.\u003c/p\u003e","description":"","filename":"MainFigure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5862113/v1/d5a2d515e1e55d8a96d7ccfb.jpg"},{"id":76844659,"identity":"4fe4ee40-9516-4440-b395-d86395b40a4d","added_by":"auto","created_at":"2025-02-21 10:42:34","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":491482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSAA1 enhance \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e resistance against formate from pneumococcal anaerobic metabolism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring \u003cem\u003eS. pneumoniae\u003c/em\u003e infection, acute pulmonary edema made alveoli become anaerobic conditions. Therefore, \u003cem\u003eS. pneumoniae \u003c/em\u003eintake SAA1 infection to improve its resistance against formate produced by pneumococcal anaerobic metabolism.\u003c/p\u003e","description":"","filename":"MainFigure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5862113/v1/e74ae366339b87a464bed029.jpg"},{"id":77747660,"identity":"d051ee32-b194-45f5-bd21-90d27ff77a5d","added_by":"auto","created_at":"2025-03-05 06:54:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3775006,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5862113/v1/b55c4958-a3ce-43cf-86dd-687edb17ce19.pdf"},{"id":76845068,"identity":"55653eee-a3c8-42c2-b403-576f64067fba","added_by":"auto","created_at":"2025-02-21 10:50:34","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":690803,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5862113/v1/1ab82f2cfe91e80b956f3d66.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Host serum amyloid A1 facilitates Streptococcus pneumoniae adaptation to acidic stress induced by pneumococcal anaerobic metabolism","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e is the most common pathogen responsible for community-acquired pneumonia, bacteremia, and sepsis.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Upon infection, \u003cem\u003eS. pneumoniae\u003c/em\u003e triggers a complex immune response characterized by the activation of both innate and adaptive immunity. This pathogen first encounters physical barriers and innate immune cells, such as alveolar macrophages, neutrophils, and dendritic cells, in the respiratory tract. Upon recognition by pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), these cells initiate a cascade of inflammatory signaling pathways.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eNeutrophils are the earliest and most abundantly recruited leukocytes in \u003cem\u003eS. pneumoniae\u003c/em\u003e infection.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e The dysfunction of neutrophils leads to the higher susceptibility of mice infected with \u003cem\u003eS. pneumonia\u003c/em\u003ee, indicating their importance in defending \u003cem\u003eS. pneumoniae\u003c/em\u003e infection.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Additionally, neutrophils also mediate phagocytosis and bacterial killing through reactive oxygen species (ROS) production and the release of antimicrobial peptides.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Although neutrophil plays a crucial role in \u003cem\u003eS. pneumoniae\u003c/em\u003e acute infection phase, the overactivation of neutrophils cause the extravasate accumulation in alveoli and excessive neutrophils even cause edematous and hemorrhagic lungs, which is a symptom in some bacterial pneumoniae patients.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e The acute pulmonary edema reduces normal oxygen movements through the lungs, thus alveoli turn to be anaerobic environments.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAs a facultative anaerobic organism, \u003cem\u003eS. pneumoniae\u003c/em\u003e lacks the cytochromes and heme containing proteins involved in aerobic respiration. Although \u003cem\u003eS. pneumoniae\u003c/em\u003e lack mechanisms for coping with oxidative stress, it is aerotolerant and showed capacity to survive under atmospheric oxygen pressure.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e The first step of pneumococcal aerobic pathway is to convert pyruvate to acetyl phosphate, which catalyzed by pyruvate oxidase (SpxB). SpxB catalyzes a two-electron reduction of O\u003csub\u003e2\u003c/sub\u003e, thereby forming the potentially damaging compound H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn addition, \u003cem\u003eS. pneumoniae\u003c/em\u003e is known to maintain a fermentative metabolism in anaerobic environments, it converts pyruvate to lactate via the lactate dehydrogenase (LDH).\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Besides lactate, most of the pyruvate can be converted to acetyl-CoA and formate by the oxygen-sensitive pyruvate formate lyase (PFL).\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Despite strong pneumococcal survival capacity in acidic stress conditions, such as the acidic environment of inflammatory foci, and tolerates lethal pH through a mechanism known as the acid tolerance response. \u003cem\u003eS. pneumoniae\u003c/em\u003e activated acidic-stress induced lysis in response to acidic environments, favoring the release of cell wall compounds, DNA and virulence factors.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBesides immunocytes, acute-phase proteins (APPs) are also rapidly synthesized by the liver in response to \u003cem\u003eS. pneuomoniae\u003c/em\u003e infection. These APPs are often triggered by pro-inflammatory cytokines like IL-6 and TNF-α.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Serum amyloid A1 (SAA1) is one such acute-phase protein, and its role during pneumococcal infections has recently garnered attention. SAA1 is known for its involvement in lipid metabolism and inflammatory responses, but its exact role in modulating the immune response during bacterial infections remains unclear, especially in Gram-positive bacterium.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e In previous studies, SAA1 was reported to bind to outer membrane protein A (OmpA) of Gram-negative bacteria, and promotes LPS clearance as well as suppresses LPS-induced inflammation and tissue injury. \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eGiven the fact that SAA1 participate in Gram-negative bacterial infection, we have interest in investigating how SAA1 works during \u003cem\u003eS. pneumoniae\u003c/em\u003e, a Gram-positive bacterial infection. In this study, we focus on elucidating the relationship between \u003cem\u003eS. pneumoniae\u003c/em\u003e infection and SAA1, and found that \u003cem\u003eS. pneumoniae\u003c/em\u003e enhance its resistance againsit formatic acidic stress by utilizing host SAA1.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eDecreased levels of SAA1 in BALF, not in serum following\u003c/b\u003e \u003cb\u003eS. pneumoniae\u003c/b\u003e \u003cb\u003echallenge in mice.\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePneumococcal infection progresses through three distinct stages, each associated with varying symptoms. First, \u003cem\u003eS. pneumoniae\u003c/em\u003e resides in the nasopharynx and patients are typically asymptomatical, Next, if \u003cem\u003eS. pneumoniae\u003c/em\u003e migrate to the lungs and patient presented with symptoms of pneumonia, leading to acute pulmonary edema. Eventually, if \u003cem\u003eS. pneumoniae\u003c/em\u003e breaches the pulmonary epithelium and enters the bloodstream, patients The patient will be diagnosed with sepsis in some severe cases.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTo investigate the host response to pneumococcal infection, we challenged mice intranasally with the \u003cem\u003eS. pneumoniae\u003c/em\u003e D39 strain (virulent serotype 2) and collected bronchoalveolar lavage fluid (BALF) and blood 24 hours post-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In this intranasal infection model, we observed a significant decrease in SAA1 levels in BALF, while C-reactive protein (CRP) levels remained stable (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). Consequently, we focused on SAA1 for subsequent experiments. Despite the reduction of SAA1 in BALF, no changes in serum SAA1 levels were detected in intranasally infected mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). This is likely because \u003cem\u003eS. pneumoniae\u003c/em\u003e did not breach the pulmonary barrier, as no bacteria were detected in the blood 24 hours post-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo examine the effects of \u003cem\u003eS. pneumoniae\u003c/em\u003e on SAA1 levels in the bloodstream, we used an intravenous infection model. Mice were challenged with the \u003cem\u003eS. pneumoniae\u003c/em\u003e D39 strain, and bacterial loads in the spleen, liver, lungs, and blood were quantified (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). \u003cem\u003eS. pneumoniae\u003c/em\u003e was successfully detected in the blood, and bacterial distribution across the spleen, liver, and lungs showed no significant differences 24 hours post-infection, confirming the successful establishment of the sepsis model (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB; Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). However, SAA1 levels in the serum remained unchanged between infection and non-infection groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results demonstrate that intranasal infection with \u003cem\u003eS. pneumoniae\u003c/em\u003e reduces SAA1 levels in BALF without affecting serum SAA1, likely due to the bacteria's inability to enter the bloodstream. In contrast, intravenous infection did not alter serum SAA1 levels, and the underlying reason requires further investigation in future studies.\u003c/p\u003e \u003cp\u003e \u003cb\u003eS. pneumoniae\u003c/b\u003e \u003cb\u003ereduces SAA1 levels via intake rather than catalysis.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate how \u003cem\u003eS. pneumoniae\u003c/em\u003e causes the reduction of SAA1 in BALF, we utilized non-infected mouse serum diluted with THY broth medium for \u003cem\u003ein vitro\u003c/em\u003e experiments, as serum contains abundant SAA1 compared to BALF. Initially, we tested whether this phenomenon is observed across various \u003cem\u003eS. pneumoniae\u003c/em\u003e strains. Serum-THY broth (10% serum, v/v) was co-cultured with two commonly used \u003cem\u003eS. pneumoniae\u003c/em\u003e strains, D39 (virulent serotype 2) and TIGR4 (virulent serotype 4). Since SAA1 is an amyloid protein with a β-sheet structure prone to aggregation, we added formic acid (0.05%, v/v), which prevents aggregation of SAA1 under conditions that bacteria remains growing (Fig. S2). Results showed that the TIGR4 strain had a stronger capacity to reduce SAA1 compared to the D39 strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Consequently, TIGR4 was selected for further investigation in studying SAA1 reduction. By measuring SAA1 levels in bacterial culture medium using ELISA, we found that formic acid can promote the SAA1 reduction by \u003cem\u003eS. pneumoniae\u003c/em\u003e. This result indicates that formic acid (0.05%, v/v) was crucial for studying SAA1 reduction with the TIGR4 strain, as no reduction occurred in its absence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrevious studies, such as those by Zhiming Wang et al., reported that \u003cem\u003eS. pneumoniae\u003c/em\u003e secretes a giant metalloprotease (IgA1 protease) to cleave host IgA1.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e We hypothesized that a pneumococcal protease might mediate SAA1 reduction. However, the addition of protease inhibitor to the culture system failed to prevent SAA1 reduction, suggesting that this process is independent of pneumococcal proteases (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). We next hypothesized that SAA1 might bind to the \u003cem\u003eS. pneumoniae\u003c/em\u003e cell membrane, a mechanism previously reported in Gram-negative bacteria. To localize SAA1, we constructed AF488-conjugated recombinant mouse SAA1 (rmSAA1) and incubated it with TIGR4. Fluorescence microscopy revealed that AF488-conjugated rmSAA1 bound to the \u003cem\u003eS. pneumoniae\u003c/em\u003e cell membrane after 2 hours of incubation, and parts of it were internalized by \u003cem\u003eS. pneumoniae\u003c/em\u003e after 3 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Additionally, enhanced fluorescence intensity were observed after permeabilization of \u003cem\u003eS. pneumoniae\u003c/em\u003e co-cultured with AF488-rmSAA1 for 3 hours (Fig. S3). These observations indicate that SAA1 reduction is due to the intake by \u003cem\u003eS. pneumoniae\u003c/em\u003e, rather than being cleaved by bacterial proteases.\u003c/p\u003e \u003cp\u003eThese results suggest that the reduction of SAA1 by \u003cem\u003eS. pneumoniae\u003c/em\u003e existing in both D39 and TIGR4 strains, but TIGR4 showing a stronger effect than D39. The process is possibly facilitated by formic acid and is independent of pneumococcal proteases. Instead, SAA1 reduction involves its binding to and subsequent internalization, and consequently intake by \u003cem\u003eS. pneumoniae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSAA1 uptake improves\u003c/b\u003e \u003cb\u003eS. pneumoniae\u003c/b\u003e \u003cb\u003eresistance to formic acidic environment.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs a 122-amino-acid protein, SAA1 is internalized by \u003cem\u003eS. pneumoniae\u003c/em\u003e and potentially utilized as a source of amino acids. In previous studies, commensal and pathogenic \u003cem\u003eE. coli\u003c/em\u003e were shown to overcome extreme acidic environments (pH\u0026thinsp;\u0026lt;\u0026thinsp;2.5) through a glutamate-dependent mechanism involving glutamate decarboxylase (GadB), which catalyzes a proton-consuming decarboxylation reaction, and the antiporter GadC, which facilitates glutamate\u003csub\u003ein\u003c/sub\u003e/γ-aminobutyrate (GABA)\u003csub\u003eout\u003c/sub\u003e exchange. This glutamate-dependent acid resistance (GDAR) system is also present in \u003cem\u003eShigella flexneri\u003c/em\u003e, \u003cem\u003eListeria monocytogenes\u003c/em\u003e, and \u003cem\u003eLactococcus lactis\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Based on this, we hypothesized that SAA1 intake might improve \u003cem\u003eS. pneumoniae\u003c/em\u003e resistance to acidic stress in anaerobic environments.\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. pneumoniae\u003c/em\u003e, like other pathogenic bacteria, must adapt to diverse and often hostile environments within the host. During acute infections, inflammatory responses lead to pulmonary edema, which impairs oxygen exchange and creates anaerobic conditions in the alveoli.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e We observed that culture medium became more acidic when \u003cem\u003eS. pneumoniae\u003c/em\u003e D39 was cultured anaerobically compared to aerobically (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Previous studies reported increased production of acetate, lactate, and formate by \u003cem\u003eS. pneumoniae\u003c/em\u003e under anaerobic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). We tested the resistance of \u003cem\u003eS. pneumoniae\u003c/em\u003e D39 to these acids and determined the maximum non-inhibitory concentrations, that are 0.05% (v/v) formic acid, 0.125% (v/v) lactic acid, and 0.083% (v/v) acetic acid (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe collected serum and measured SAA1 levels using ELISA, then seperated serum into serum \u003csup\u003ehigh\u0026minus;SAA1\u003c/sup\u003e (400\u0026thinsp;~\u0026thinsp;1000 \u0026micro;g/mL SAA1) and serum \u003csup\u003elow\u0026minus;SAA1\u003c/sup\u003e (10\u0026thinsp;~\u0026thinsp;20 \u0026micro;g/mL SAA1) groups. To investigate whether SAA1 facilitates \u003cem\u003eS. pneumoniae\u003c/em\u003e resistance to acidic stress, we cultured \u003cem\u003eS. pneumoniae\u003c/em\u003e D39 in lethal concentrations of formic (0.075%, v/v), lactic (0.1875%, v/v), and acetic (0.125%, v/v) acids with 10% serum containing either serum \u003csup\u003ehigh\u0026minus;SAA1\u003c/sup\u003e or serum \u003csup\u003elow\u0026minus;SAA1\u003c/sup\u003e. Both serum \u003csup\u003ehigh\u0026minus;SAA1\u003c/sup\u003e and serum \u003csup\u003elow\u0026minus;SAA1\u003c/sup\u003e enhanced \u003cem\u003eS. pneumoniae\u003c/em\u003e D39 resistance to lethal formic acid (0.075%, v/v), with serum \u003csup\u003ehigh\u0026minus;SAA1\u003c/sup\u003e promoting better bacterial growth after 10 hours compared to serum \u003csup\u003elow\u0026minus;SAA1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). However, SAA1 contributed minimally to resistance against acetic and lactic acids (Fig. S4A, S4B). To further confirm that SAA1 uptake enhances resistance to formic acid, we cultured \u003cem\u003eS. pneumoniae\u003c/em\u003e with AF488-rmSAA1 and used propidium iodide to stain dead cells. Propidium iodide, which penetrates the compromised membranes of dead bacteria, binds to DNA and fluoresces.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Under fluorescence microscopy, few green bacteria (indicating AF488-rmSAA1 intake) were stained with propidium iodide after 2 hours of exposure to lethal formic acid (0.25%,v/v) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD; Fig. S5). This result aligns with the growth curve findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, E), leading to the conclusion that SAA1 intake improves \u003cem\u003eS. pneumoniae\u003c/em\u003e resistance to formic acid stress.\u003c/p\u003e \u003cp\u003eIt should be noted that serum \u003csup\u003elow\u0026minus;SAA1\u003c/sup\u003e was obtained from untreated 8-week-old C57BL/6 mice, while serum \u003csup\u003ehigh\u0026minus;SAA1\u003c/sup\u003e was derived from mice treated with intranasal PBS administration, which significantly elevated serum SAA1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B, C). It is an original PBS-based intranasal administration method to induce SAA1 increases in serum. This innovative approach provides a convenient platform for studying the role of SAA1 in various bacterial species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results indiciate that SAA1 intake by \u003cem\u003eS. pneumoniae\u003c/em\u003e enhances bacterial resistance to formic acid stress in anaerobic environments. High SAA1 concentrations in serum significantly boost this resistance, while the effect on resistance to acetic and lactic acids is minimal (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDuring \u003cem\u003eS. pneumoniae\u003c/em\u003e infection, the bacterium triggers a complex immune response involving both innate and adaptive immunity. Neutrophils, as the earliest and most abundant leukocytes recruited to the site of infection, play a critical role. Meanwhile, dendritic cells process bacterial antigens and present them to T cells, initiating an adaptive immune response. Among these, T helper 17 (Th17) cells, a subset of CD4\u0026thinsp;+\u0026thinsp;T cells, are pivotal in producing IL-17, which promotes neutrophil recruitment and enhances mucosal defense against \u003cem\u003eS. pneumoniae\u003c/em\u003e. Additionally, B cells generate specific antibodies that facilitate opsonization, neutralize the bacterial capsule, and promote pathogen clearance through complement activation. The development of memory B cells and durable antibodies is particularly important for preventing reinfection and improving vaccine efficacy.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eWhile the roles of various immune cells in \u003cem\u003eS. pneumoniae\u003c/em\u003e infection have been extensively studied, there is limited understanding of how acute-phase proteins (APPs) function alone or in coordination with immune cells during such infections. C-reactive protein (CRP), for instance, is known to bind phosphocholine on the bacterial surface, promoting opsonization and complement-mediated clearance.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Although widely studied as a biomarker of bacterial infection and inflammation, CRP's specific role in pneumococcal immunity highlights the importance of systemic acute-phase responses. To date, the function of SAA1 has been explored mainly in Gram-negative bacterial infections. For example, SAA1 has been reported to bind to outer membrane protein A (OmpA) of Gram-negative bacteria, promoting lipopolysaccharide (LPS) clearance and suppressing LPS-induced inflammation and tissue injury.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this study, we investigated the role of SAA1 in \u003cem\u003eS. pneumoniae\u003c/em\u003e infection. We observed that nasal infection with \u003cem\u003eS. pneumoniae\u003c/em\u003e led to a reduction of SAA1 in mice BALF (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Similarly, when \u003cem\u003eS. pneumoniae\u003c/em\u003e D39 and TIGR4 strains were co-cultured with serum-THY broth (10% serum, v/v) for 12 hours \u003cem\u003ein vitro\u003c/em\u003e, SAA1 levels decreased, with the TIGR4 strain showing a stronger capacity to reduce SAA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Consequently, we selected the TIGR4 strain as a model to study the mechanism underlying this reduction. Since \u003cem\u003eS. pneumoniae\u003c/em\u003e produces IgA1 protease, we hypothesized the presence of an SAA1-specific protease. However, adding protease inhibitors to the culture system failed to prevent SAA1 reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Instead, we observed that AF488-rmSAA1 started to be internalized by \u003cem\u003eS. pneumoniae\u003c/em\u003e after three hours of co-culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). These findings suggest that \u003cem\u003eS. pneumoniae\u003c/em\u003e reduces SAA1 levels in BALF by actively intaking SAA1.\u003c/p\u003e \u003cp\u003eInterestingly, we found that formic acid is necessary for SAA1 intake by \u003cem\u003eS. pneumoniae\u003c/em\u003e TIGR4, though the underlying mechanism remains unclear. Initially, we added formic acid (0.05%,v/v) to the culture system to prevent SAA1 aggregation. The results suggest that formic acid may activate \u003cem\u003eS. pneumoniae\u003c/em\u003e to internalize SAA1, potentially aiding its adaptation to formic acid stress. However, no receptor or protein capable of sensing formic acid concentrations has been identified in either prokaryotes or eukaryotes. Formic acid is commonly used as a preservative and antibacterial agent in livestock feed, and it is also produced as a short-chain fatty acid (SCFA) by intestinal microorganisms during metabolism.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Although acetate, propionate, and butyrate constitute the majority of SCFAs in the human colon, formic acid is present in smaller amounts and may be associated with methanogenesis or elevated during inflammation. Its physiological and pathological roles remain largely unexplored, highlighting the need for further investigation.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOne challenge in studying SAA1 function is the high cost of recombinant SAA1 due to the difficulties in purifying it from \u003cem\u003eE. coli\u003c/em\u003e expression systems, stemming from its fibrillation and amphiphilic properties.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Previous methods for inducing SAA1 production in mice, such as subcutaneous silver nitrate injections or intranasal LPS administration, are either hazardous or costly and can interfere with experimental outcomes by inducing antibody production.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e In this study, we developed a cost-effective and stable method to prepare SAA1-abundant serum by intranasal administration of PBS in male C57BL/6 mice. This approach successfully induced SAA1 production, although the underlying mechanism remains unclear. We collected serum with high SAA1 levels (serum \u003csup\u003ehigh\u0026minus;SAA1\u003c/sup\u003e) from PBS-treated mice and serum with low SAA1 levels (serum \u003csup\u003elow\u0026minus;SAA1\u003c/sup\u003e) from untreated mice. This method enabled us to compare the effects of SAA1 using serum samples first and then confirm the results with recombinant SAA1.\u003c/p\u003e \u003cp\u003eThe glutamate-dependent acid resistance (GDAR) system, found in \u003cem\u003eShigella flexneri\u003c/em\u003e, \u003cem\u003eListeria monocytogenes\u003c/em\u003e, and \u003cem\u003eLactococcus lactis\u003c/em\u003e, facilitates bacterial adaptation to acidic stress.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Here, we investigated whether SAA1 could enhance \u003cem\u003eS. pneumoniae\u003c/em\u003e resistance to acidic stress. During pneumonia, \u003cem\u003eS. pneumoniae\u003c/em\u003e transitions from the nasopharynx to the lungs, where fluid accumulation and oxygen reduction create anaerobic conditions. Previous studies have shown that \u003cem\u003eS. pneumoniae\u003c/em\u003e produces acetate, lactate, and formate under anaerobic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). We found that the pH of \u003cem\u003eS. pneumoniae\u003c/em\u003e cultures was lower under anaerobic conditions than aerobic ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Due to technical limitations, we could not identify specific metabolites but selected acetate, lactate, and formate as representative byproducts. Using serum \u003csup\u003ehigh\u0026minus;SAA1\u003c/sup\u003e and serum \u003csup\u003elow\u0026minus;SAA1\u003c/sup\u003e, we observed that \u003cem\u003eS. pneumoniae\u003c/em\u003e D39 displayed better growth in lethal concentrations of formic acid (0.075%, v/v) when co-cultured with serum \u003csup\u003ehigh\u0026minus;SAA1\u003c/sup\u003e compared to serum \u003csup\u003elow\u0026minus;SAA1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, e). This finding was further confirmed using AF488-rmSAA1 and fluorescence microscopy, which demonstrated that SAA1 intake enhances \u003cem\u003eS. pneumoniae\u003c/em\u003e resistance to formic acid stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eIn conclusion, our study reveals a novel adaptation strategy employed by \u003cem\u003eS. pneumoniae\u003c/em\u003e to cope with formic acid stress by internalizing host SAA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This interaction between an acute-phase protein and a bacterial pathogen enhances the pathogen\u0026rsquo;s ability to resist acidic conditions. By utilizing SAA1, an acute-phase protein associated with immune defense and inflammation, \u003cem\u003eS. pneumoniae\u003c/em\u003e gains a survival advantage in acidic environments, improving its fitness during infection and evading metabolic stress. This discovery expands our understanding of pneumococcal survival strategies and highlights the importance of host-pathogen interactions in shaping bacterial adaptation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e Our research complied with all of the relevant ethical regulations. All of the animal procedures were conducted according to the protocols approved by Animal Care and Use Committee of Osaka University Graduate School of Dentistry (authorization number 04-018-0).\u003c/p\u003e \u003cp\u003eAll methods are reported in accordance with the ARRIVE guidelines (Animal Research: Reporting of \u003cem\u003eIn Vivo\u003c/em\u003e Experiments) to ensure rigorous and transparent reporting of animal research (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eBacterial culture\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e D39 (serotype 2 clinical isolate) and TIGR4 (virulent serotype 4 clinical isolate) strains were cultured in Todd-Hewitt broth (Becton, Dickinson and Company [BD], New Jersey, U.S.) supplemented with 0.2% yeast extract (BD) (THY broth) at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e\n\u003ch3\u003eAnimal infection experiments\u003c/h3\u003e\n\u003cp\u003eFemale Slc:ICR mice at 6 to 7 weeks old (SLC Japan, Inc., Shizuoka, Japan) were used as animal infection model in this study. The \u003cem\u003eS. pneumoniae\u003c/em\u003e D39 strain was grown to the early-exponential phase (OD\u003csub\u003e600\u003c/sub\u003e of 0.1) and then washed with and resuspended in PBS.\u003c/p\u003e \u003cp\u003eFor intranasal infection experiments, each mouse was intraperitoneally given 250 \u0026micro;L of anaesthetic consisting of 20 \u0026micro;L midazolam (Takeda Pharmaceuticals, Osaka, Japan), 7.5 \u0026micro;L domitor (ZENOAQ, Fukushima, Japan), 25 \u0026micro;L vetorphale (Meiji Animal Health Co., Ltd., Kumamoto, Japan) and 197.5 \u0026micro;L PBS solution, then mice were given to mice by administration of 4 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU in 20 \u0026micro;L PBS. Blood and BALF were collected from mice 24 hours post intranasal infection after mice were euthanized by intraperitoneal injection of 300 \u0026micro;L pentobarbital sodium solution (20 mg/mL, Product mumber: P0776, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan). BALF were collected by slowly drawing the injected PBS back into the syringe and the serum were collected from blood by centrifugation (8,500 rpm, 15 minutes, 4 ˚C).\u003c/p\u003e \u003cp\u003eIn intravenous infection experiments, bacteria were introduced into mice by administration of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU in 100 \u0026micro;L PBS. For quantification of bacterial colonization in the lung, liver, spleen and blood, mice were euthanized 24 hours after \u003cem\u003eS. pneumoniae\u003c/em\u003e infection by intraperitoneal injection of 300 \u0026micro;L pentobarbital sodium solution (20 mg/mL, Product mumber: P0776, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) and then lung, liver, spleen tissues and blood were immediately collected. Tissue homogenates and blood were serially diluted and plated on THY agar plates and then incubated at 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. After 24 hours incubation, colony-forming unit (CFU) per weight of organ tissues (gram) or volume of blood (mL) was calculated.\u003c/p\u003e\n\u003ch3\u003eQuantification of SAA1 and C-reaction protein (CRP) using ELISA\u003c/h3\u003e\n\u003cp\u003eAfter the collection of serum and BALF from mice, SAA1/2 and CRP quantification was performed by ELISA following the manufacturer's protocols described in mouse serum amyloid A ELISA Kit (SAA; Cat# KMA0021, Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, U. S.) and mouse CRP ELISA Kit (Cat# EM20RB, Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, U. S.).\u003c/p\u003e \u003cp\u003eIn SAA1 reduction \u003cem\u003ein vitro\u003c/em\u003e experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C), we utilized absorbance intensity (OD\u003csub\u003e450\u003c/sub\u003e) instead of quantification to determine SAA1 levels.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of serum \u003csup\u003ehigh\u0026minus;SAA1\u003c/sup\u003e and serum \u003csup\u003elow SAA\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e\u003c/h2\u003e \u003cp\u003eMale C57BL/6J mice at 7 to 8 weeks old (SLC Japan, Inc., Shizuoka, Japan) were used as source of serum \u003csup\u003ehigh\u0026minus;SAA1\u003c/sup\u003e and serum \u003csup\u003elow\u0026minus;SAA1\u003c/sup\u003e. 20 \u0026micro;L of 1x phosphate-buffered saline (PBS) were given to mice intranasally. After 24 hours, mice were euthanized by intraperitoneal injection of 300 \u0026micro;L pentobarbital sodium solution (20 mg/mL, Product mumber: P0776, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), and cardiac puncture was performed to collect blood. The serum was collected from blood by centrifugation (8,500 rpm, 15 minutes, 4 ˚C). SAA1 concentration of each sample were determined by mouse serum amyloid A ELISA Kit (SAA; Cat# KMA0021, Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, U. S.). This method was confirmed to significantly and stably increase the serum SAA1 level. Thus, serum \u003csup\u003ehigh\u0026minus;SAA1\u003c/sup\u003e were collected from PBS-intranasal administration and serum \u003csup\u003elow\u0026minus;SAA1\u003c/sup\u003e were collected from mice without PBS-intranasal administration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCo-culture of\u003c/b\u003e \u003cb\u003eS. pneumoniae\u003c/b\u003e \u003cb\u003ewith SAA1-abundant serum\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe SAA1-abundant serum from PBS-intranasal administration C57BL/6J mice and the SAA1 concentration were measured by mouse serum amyloid A ELISA Kit (SAA; Cat# KMA0021, Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, U. S.). \u003cem\u003eS. pneumoniae\u003c/em\u003e D39 strain or TIGR4 strain was grown to the early-exponential phase (OD\u003csub\u003e600\u003c/sub\u003e of 0.1). Then, 5 \u0026micro;L of bacterial culture were added into 20 \u0026micro;L serum and 175 \u0026micro;L THY broth (containing final 0.05% formic acid, v/v) in a 96-well plate and incubated in 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 12 hours. To test whether SAA1 reduction was protease-dependent, protease inhibitor (cOmplete\u0026trade;, EDTA-free Protease Inhibitor Cocktail, Roche, Swiss) was dissolved and used following its instruction for use.\u003c/p\u003e \u003cp\u003eAfter incubation, the supernatent of bacterial culture were collected after centrifugation (12,000 rpm, 10 minutes, 4 ˚C) and the remaining SAA were measured using mouse serum amyloid A ELISA Kit (SAA; Cat# KMA0021, Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, U. S.).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAF488-conjugated recombinant SAA1 intake by\u003c/b\u003e \u003cb\u003eS. pneumoniae\u003c/b\u003e \u003cb\u003eobservation using fluorescence microscope\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRecombinant mouse SAA1(rmSAA1) (Cat# 2948-SA-025, R\u0026amp;D Systems, Minneapolis, U.S.) were conjugated with AF488 following the protocol described in Alexa Fluor\u0026reg; 488 Conjugation Kit (Fast) - Lightning-Link\u0026reg; (Abcam, Cat# ab236553, Abcam Limited, Cambridge, U. K.). \u003cem\u003eS. pneumoniae\u003c/em\u003e TIGR4 strain was grown to the early-exponential phase (OD\u003csub\u003e600\u003c/sub\u003e of 0.1) and 1 mL of bacteria were collected and resuspended with 50 \u0026micro;L PBS containing AF488-rmSAA1 (final concentration\u0026thinsp;=\u0026thinsp;0.25 \u0026micro;g/mL) with existence 0.05% formic acid (concentration that not inhibiting pneumococcal growth) or 0.25% formic acid (lethal concentration). Then, bacteria were incubated in 5% CO\u003csub\u003e2\u003c/sub\u003e incubator, 5 \u0026micro;L of bacteria were collected after 1 hour, 2 hours and 3 hours, then were observed using fluorescence microscope (KEYENCE BZ-X, Osaka, Japan).\u003c/p\u003e \u003cp\u003eIn Fig. S3, we collected the bacterial pellet at 3 hours post incubation through centrifugation (12,000 rpm, 10 minutes, 4 ˚C) and then added 0.05% Triton X-100 into bacterial pellet to permeabilize bacteria, then its fluorescence intensity (Ex/Em\u0026thinsp;=\u0026thinsp;485/535) was measured after permeabilization for 0, 0.5, 1 hour.\u003c/p\u003e \u003cp\u003eIn Fig. S4, we cultured \u003cem\u003eS. pneumoniae\u003c/em\u003e in 0.25% formic acid together with AF488-rmSAA1 (final concentration\u0026thinsp;=\u0026thinsp;0.25 \u0026micro;g/mL). After 2 hours incubation, Propidium Iodide was added to bacteria (final concentration\u0026thinsp;=\u0026thinsp;1 \u0026micro;g/mL) and incubated at 37 ˚C for 15 minutes. After incubation, bacteria were washed twice and resuspended with PBS. Then, the bacteria were observed under fluorescence microscope (KEYENCE BZ-X, Osaka, Japan).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003epH measurement of bacterial culture under aerobic and anaerobic environment\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e D39 were added in a 24-well plate, then cultured in aerobic (5% CO\u003csub\u003e2\u003c/sub\u003e incubator) and anaerobic environment created by using AnaeroPack-Anaero (MITSUBISHI GAS CHEMICAL Co. Inc, Japan). The supernatant of bacterial cultures was collected at different time points (1, 3, 5, 7, 9 hours). 50 \u0026micro;L of 0.04% (w/v) phenol red broth (Cat# 165\u0026ndash;01121, FUJIFILM Wako Pure Chemical Corporation, Japan) were mixed with 950 \u0026micro;L bacterial supernatant collected at each point was measured the absorbance at 560 nm. A calibration curve was determined in phenol red broth which adjusted to pH values ranging from 4 to 10. For THY broth, culture supernatant at each point was supplemented with 0.04% phenol red and the absorbance was measured at 550 nm. A calibration curve was determined in THY broth which was supplemented with 5 mg/mL phenol red and adjusted to pH values ranging from 3 to 10.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBacterial growth curve in lethal formic, lactic and acetic acid concentration.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eS. pneumoniae\u003c/em\u003e D39 strain was grown to the early-exponential phase (OD\u003csub\u003e600\u003c/sub\u003e of 0.1). Then, 5 \u0026micro;L of bacterial culture were added into 20 \u0026micro;L serum \u003csup\u003ehigh\u0026minus;SAA1\u003c/sup\u003e and serum \u003csup\u003elow\u0026minus;SAA1\u003c/sup\u003e and 175 \u0026micro;L THY broth (containing 0.125% acetic, 0.1875% lactic, and 0.075% formic acid, v/v) in a 96-well plate and incubated in 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 12 hours. The OD\u003csub\u003e600\u003c/sub\u003e of each well was recorded by microplate reader (Infinite\u0026reg; M Plex, Tecan, Swiss).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll results were statistically analysed using the Student's \u003cem\u003et-\u003c/em\u003etest in Excel (v. 16.81; Microsoft, Redmond, WA, USA). Differences were considered statistically significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData visualization\u003c/h2\u003e \u003cp\u003eHiplot (ORG) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hiplot.org\u003c/span\u003e\u003cspan address=\"https://hiplot.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), a comprehensive and easy-to-use web service for boosting publication-ready biomedical data visualization, was used to visualize the following data: Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-E; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, C; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C; Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The other data were created by Excel (v. 16.81; Microsoft, Redmond, WA, USA) and modified by Keynote (v. 14.1; Apple Inc., California, U. S.)\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the OpenBiox community and the Hiplot team (https://hiplot.org) for providing technical assistance and valuable tools for data analysis and visualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by JST SPRING (grant number JPMJSP2138). The funders had no role in the study design, data collection or analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eW.G. and S.K. designed the study. W.G., M.O., and K.M. performed the experiments. T.S. and Y.H. gave their valuable and helpful suggestion. W.G. wrote the original manuscript. All authors reviewed and edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated in this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll mouse experiments were conducted using a protocol approved by the Animal Care and Use Committee of Osaka University Graduate School of Dentistry (authorization number 04-018-0).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAustrian, R. 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Med.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e (6), 5275\u0026ndash;5282 (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Serum amyloid A1, Streptococcus pneumoniae, formic acid","lastPublishedDoi":"10.21203/rs.3.rs-5862113/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5862113/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSerum amyloid A1 (SAA1), an acute-phase protein, exhibits a decreased level in bronchoalveolar lavage fluid (BALF) following nasal \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e infections. However, the function of SAA1 in relation to \u003cem\u003eS. pneumoniae\u003c/em\u003e remains to be unclear. In this study, we investigate whether \u003cem\u003eS. pneumoniae\u003c/em\u003e utilized SAA1 for its survival in host or not.\u003c/p\u003e \u003cp\u003eWe explored the function of SAA1 in relation to \u003cem\u003eS. pneumoniae\u003c/em\u003e by initially comparing serum with high SAA1 levels to serum with low SAA1 levels. We then validated our findings using recombinant SAA1. In this research, we developed an original PBS-based intranasal administration method to induce SAA1 increases in serum. This innovative approach provides a convenient platform for studying the role of SAA1 in various bacterial species.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e experiments showed that when serum-THY broth was incubated with \u003cem\u003eS. pneumoniae\u003c/em\u003e, a similar reduction in SAA1 was observed, which was proved to be independent of protease activity. Intake of AF488 (green-fluorescent probe)-conjugated recombinant SAA1 by \u003cem\u003eS. pneumoniae\u003c/em\u003e was observed using fluoresence microscope. Then, we proved that intake of SAA1 enhanced \u003cem\u003eS. pneumoniae\u003c/em\u003e resistance against formate, a key metabolite of its anaerobic metabolism.\u003c/p\u003e \u003cp\u003eThis study is the first to reveal the relationship between SAA1 and a Gram-positive bacterium. We demonstrate that \u003cem\u003eS. pneumoniae\u003c/em\u003e exploits host acute-phase proteins to enhance its survival in formic acidic environments. These findings expand our understanding of bacterial survival strategies, highlighting how pathogens utilize host-derived components to adapt to hostile conditions.\u003c/p\u003e","manuscriptTitle":"Host serum amyloid A1 facilitates Streptococcus pneumoniae adaptation to acidic stress induced by pneumococcal anaerobic metabolism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-21 10:42:29","doi":"10.21203/rs.3.rs-5862113/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"64353649-18c2-414e-9027-fa98ae3c6dee","owner":[],"postedDate":"February 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":44618731,"name":"Biological sciences/Microbiology/Bacteria/Bacterial host response"},{"id":44618732,"name":"Biological sciences/Microbiology/Bacteria/Bacterial physiology"},{"id":44618733,"name":"Biological sciences/Microbiology/Bacteria/Bacterial synthetic biology"}],"tags":[],"updatedAt":"2025-03-05T06:54:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-21 10:42:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5862113","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5862113","identity":"rs-5862113","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}