Long-term control of Salmonella after transient T3SS-2 inhibition

Long-term control of Salmonella after transient T3SS-2 inhibition | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Long-term control of Salmonella after transient T3SS-2 inhibition View ORCID Profile Alaa Alhayek , Beatrice Claudi , Sila Nizam , Helen Atassi , View ORCID Profile Dirk Bumann doi: https://doi.org/10.1101/2025.10.30.685576 Alaa Alhayek 1 Biozentrum, University of Basel , Basel, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alaa Alhayek Beatrice Claudi 1 Biozentrum, University of Basel , Basel, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sila Nizam 1 Biozentrum, University of Basel , Basel, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Helen Atassi 1 Biozentrum, University of Basel , Basel, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dirk Bumann 1 Biozentrum, University of Basel , Basel, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Dirk Bumann For correspondence: dirk.bumann{at}unibas.ch Abstract Full Text Info/History Metrics Preview PDF Abstract Anti-virulence approaches are promising alternatives for traditional antibiotics to control bacterial infections. Several inhibitors show impressive activities in animal infection models, but the relative contribution of specific virulence inhibition vs off-target effects on both the bacteria and host remain unclear. Here, we developed Salmonella with switchable virulence by putting the type 3 secretion system-2 (T3SS-2) which is essential for systemic virulence, under the control of doxycycline. In infected mice given low-dose doxycycline in drinking water, the strain showed normal fitness and virulence. Doxycycline withdrawal shut down T3SS-2, arrested Salmonella replication and resolved disease symptoms. After ten days of T3SS-2 inhibition, reintroducing doxycycline restored replication, but bacterial loads remained stable, indicating strengthened host immunity. These effects were comparable to treatment with fluoroquinolone antibiotics, a highly effective therapy for human systemic salmonellosis. Thus, selective T3SS-2 inhibition may offer a suitable alternative for controlling invasive Salmonella infections. Main Bacterial infections remain a major threat to human health. Most pathogens have evolved resistance to available antibiotics 1 . There has also been a long gap in antibiotic discovery, with few novel antibiotics coming 2 . However, the increasing understanding of bacterial pathogenesis has revealed potential non-antibiotic approaches. One of these is virulence inhibition which disarms pathogens in the interaction with the host immune system rather than killing them. This minimizes collateral damage to the host microbiome and slows resistance development 3 , 4 . This approach has a long history of success, from early toxin-neutralizing ( e . g ., tetanus) to modern monoclonal antibodies 5 , 6 and small-molecule inhibitors 4 . Many of these small molecules show potent in vitro activity, but variable in vivo activity 7 – 9 . Further development of the most promising leads also remains scarce. Open questions include: can virulence inhibitors clear infections on their own? Can such inhibitor lead to long-term control of infections without relapsing disease after termination of virulence inhibition? One widely studied virulence system that is suitable to address these questions, is type III secretion system (T3SS), in which an injectosome translocates virulence factors from the bacterial cytosol into host cells 10 . T3SS is essential for virulence of diverse Gram-negative pathogens, including Pseudomonas aeruginosa, Shigella, Yersinia , and Salmonella 11 , 12 . Among these, Salmonella enterica is a major cause of mortality worldwide and becomes increasingly untreatable 13 , 14 motivating the WHO to classify it as a priority pathogen for antimicrobial discovery 15 . Salmonella encodes two T3SSs: T3SS-1, which mediates epithelial invasion, and T3SS-2, which supports intracellular growth in macrophages 16 . Salmonella mutants lacking T3SS-2 activity are avirulent for systemic disease in mice and humans 17 , 18 . T3SS-2 has therefore emerged as a promising target for anti-virulence development. However, a large part of the effector proteins translocated through T3SS-2 have anti-inflammatory activities 19 , so abrupt inhibition could exacerbate inflammation. T3SS-2 inhibitors have been reported 7 , 20 , 21 . Some of them reduce mortality in mouse infection models 22 – 25 (and human urinary-tract infections 26 ), implying that T3SS-2 activity is required continuously throughout infection. However, some of the inhibitors are likely confounded with off-target effects both in the bacteria and in mammalian host cells 22 , 25 . Thus, the immediate and long-term impact of transient T3SS-2 inhibition in highly infected mice remains unclear. Establishing and validating a switchable anti-virulence approach in Salmonella To determine the impact of anti-virulence treatments on Salmonella control in vivo, we developed a switchable system that activates or inactivates T3SS-2 without affecting other aspects of Salmonella biology or the host ( Fig. 1a ). T3SS-2 depends on the transcription factor SsrB 27 . We inactivated ssrB by multiple STOP codons across the gene to reduce polar effects on downstream ssrA . This abolished the activity of a SsrB-dependent P sifB - gfp transcriptional fusion encoding an unstable variant of GFP (GFP-OVA 28 , 29 ) in T3SS-2 inducing conditions 30 . We complemented the ssrB mutant with a doxycycline (DOX)-inducible P tetA - ssrB expression cassette. We used DOX as inducer because of its bioavailability in mice 31 and stability in acidified drinking water 32 . In presence of 0.125 mg/L DOX (well below the minimum inhibitory concentration, 1-2 mg/L), P sifB - gfp regained full activity ( Fig. S1b ). However, there was also substantial activity even in absence of DOX, suggesting leaky ssrB expression ( Fig. S1b ). We optimized the dynamic range of switchable ssrB using an inefficient ribosomal binding site, resulting in low baseline and full DOX-induced activity of P sifB - gfp ( Fig. 1b ). We also included a constitutive mCherry expression cassette ( Fig. S1a ) to detect all Salmonella regardless of P tetA - ssrB activity. We called this construct pVIR-ON/OFF. Download figure Open in new tab Figure S1. Assessment of doxycycline dosing in mice. (a) Plasmid map of the switchable-virulence construct. A constitutive P ybaJ -mCherry cassette labels all Salmonella irrespective of P tetA -ssrB activity. (b) DOX dependence of P sifB -gfp activity in initial not optimized construct. Autofluorescence of a (WT, wild-type) Salmoenlla (gray) and induced P sifB - gfp activity in WT is shown in (orange). Pooled data from three mice per group. (c) Green fluorescence of P sifB -gfp in Salmonella with inactivated ssrB in spleen of infected mice. Autofluorescence of a (WT, wild-type) Salmoenlla (gray) and induced P sifB - gfp activity in WT is shown in (orange). Pooled data from three mice per group. (d) Continuous P sifB -gfp activity in mice at day 6 post-infection, after receiving 0.1 mg/mL DOX for 5 days and 1 day without DOX. Autofluorescence of a (WT, wild-type) Salmoenlla (gray) and induced P sifB - gfp activity in WT is shown in (orange). Pooled data from three mice per group. (e) Body weight change (% of initial) over time. DOX was provided from day 1 to day 3, withdrawn until day 14, then re-administered until day 21. Data represent means of three to seven mice per group and their standard error of the mean. (f) Plasmid map of P tetA -ssrB with constitutive timer bac expression. Download figure Open in new tab Figure S2. Gating strategy for in vitro and in vivo flow cytometry data. (a) Identification of single bacteria by FSC vs SSC, followed by gating on constitutive mCherry + events to define all Salmonella with the construct. (b) Gate used to identify Salmonella with high constitutive mCherry expression in spleen homogenates. Download figure Open in new tab Figure 1. Validation of a switchable anti-virulence system in Salmonella . (a) Schematic description of the switchable system controlling T3SS-2. (b) In vitro validation of switchable anti-virulence, (DOX, Doxycycline). Autofluorescence of a (WT, wild-type) Salmoenlla (gray) and induced P sifB - gfp activity in WT is shown in (orange). Pooled data from two-independent experiments. (c) Green fluorescence of the switchable virulence Salmoenlla in mice receiving 0.01 mg/mL DOX in drinking water. Induced PsifB-gfp (orange) is high with DOX; after 3 days of DOX withdrawal, PsifB-gfp returns to baseline (blue). Autofluorescence of a (WT, wild-type) Salmoenlla (gray) and induced P sifB - gfp activity in WT is shown in (orange). Pooled data from three mice per group. (d) Green fluorescence after 6 days without DOX and one day of re-exposure. Autofluorescence of a (WT, wild-type) Salmoenlla (gray) and induced P sifB - gfp activity in WT is shown in (orange). (e) Time course of splenic GFP signal under DOX until day 3 and from day 14; pooled data from three independently infected mice. Autofluorescence of a (WT, wild-type) Salmoenlla (gray) and induced P sifB - gfp activity in WT is shown in (orange). Data for individual mice are shown in panel f. (f) Median green fluorescence intensity (MFI) for data shown in panel e. Each circle represents one mouse; the bar is the mean. The dashed line represents the MFI of wild-type Salmonella without gfp . To test pVIR-ON/OFF in vivo, we provided DOX in drinking water to mice. We used mice with functional SLC11A1 to avoid sever immunosuppression 33 , 34 . Previous studies used 0.15 to 2 mg/mL for inducing microbial gene expression from P tetA 32 , 35 . To minimize potential antimicrobial effects of DOX on Salmonella , we initially used 0.1 mg/mL DOX from 1d prior to and throughout infection. Mice infected with Salmonella /pVIR-ON/OFF ( S -VIR) under these conditions developed symptoms comparable to mice infected with wild-type Salmonella without DOX. Activity of P sifB - gfp in spleen at d5 post infection were also comparable ( Fig S1d ). Conversely, infection of mice with S -VIR without DOX caused no disease symptoms (data not shown), little bacterial growth as reported before 36 , and only baseline P sifB - gfp activity similar to data for a Salmonella ssrB mutant ( Fig S1c ). Thus, S -VIR provided conditional SPI-2 activity, fitness, and virulence in vivo. To simulate an anti-virulence treatment, we infected DOX-treated mice with S -VIR and withdrew DOX at 5d post-infection. However, at d6 we observed still high P sifB - gfp activity ( Fig S1d ), suggesting ongoing T3SS-2-activity. This could be due to slow elimination of DOX from tissue 31 and/or stability of previously made SsrB. We thus further reduced the DOX concentration to 0.01 mg/mL and withdrew DOX already at d3 post-infection. Under these conditions, disease symptoms and spleen S -VIR loads still increased until d6 indicating high fitness and virulence, but P sifB - gfp activity declined to baseline ( Fig. 1e ). When these mice were exposed again to 0.01 mg/mL DOX in the drinking water, S -VIR regained full activity ( Fig. 1d & f ). These data demonstrated that S -VIR was a fully functional switchable virulence construct. Disease relapse but CFU control post-anti-virulence To determine the impact of virulence suppression and regain, we infected DOX-treated mice with S -VIR, withdrew DOX at d3 post-infection, and re-administered DOX from d14 modulating T3SS-2 activity accordingly ( Fig 1f & 2a). Disease symptoms and bacterial loads in spleen increased until d6 ( Fig 2a & b ). In the absence of DOX, symptoms resolved and bacterial loads remained constant, confirming the suppression of virulence without eradicating the bacteria. Constant bacterial loads could reflect a replication arrest and no host-mediated killing, or a dynamic equilibrium between ongoing replication and killing. To distinguish between these alternatives, we combined P tetA - ssrB with timer bac expression ( Fig S1f ) which reports bacterial replication rates via fluorescence spectral shifts 34 , 37 , 38 . TIMER bac revealed normal Salmonella replication at d3 in presence of DOX, but replication arrest at d10 and d14 in absence of DOX ( Fig 2c ). Thus, suppression of SPI-2 stopped replication in vivo without enabling host-mediated killing, consistent with the phenotype of SPI-2-deficient mutants 36 , 39 . Download figure Open in new tab Figure 2. In vivo impact of virulence modulator. (a) Clinical scores and (b) spleen bacterial loads in mice infected with the switchable-virulence strain (S-VIR). (DOX, Doxycycline) was given from day 1 to day 3, withdrawn until day 14, then re-administered until day 21, (WT, wild-type). (c) Salmonella division rates. The histograms represent pooled three to seven mice. Data for each individual mice are shown in the inset. Each circle represents one mouse; the bars show the mean. (d) Salmonella burdens in spleen in mice anti-CD4–treated versus isotype control mice. Each circle represents an individual mouse; the bars show the geometric means. (e–f) Effects of enrofloxacin treatment on clinical scores (e) and spleen CFU (f). (g) Timeline of combined treatment with ciprofloxacin (CIP) and DOX to modulate T3SS-2 activity. (h) Comparison of antibiotic alone versus combined treatment (CIP + T3SS-2 OFF). Each circle represents an individual mouse; the line shows the geometric mean. (i) Proposed working model for anti-virulence treatment. Post anti-virulence replication restarted, but host killing leads to partial control. Upon re-administration of DOX from d14, restarted of Salmonella replication ( Fig. 2b & 2c ). However, bacterial loads remained constant, suggesting enhanced Salmonella killing by the host. These enhanced host activities were associated with re-appearance of increasing symptoms. Thus, virulence suppression mitigated disease symptoms and stopped Salmonella growth. During this time, the host gained the capability to control Salmonella net growth even after restoring virulence, but this was insufficient to eradicate the bacteria and to prevent relapsing disease ( Fig 2i ). Minimal CD4 + T-cell role in post–T3SS-2 protection CD4 + T-cells contributes to protective immunity against Salmonella after vaccination or during chronic infection 40 – 43 . To test their contribution to post–anti-T3SS-2 control, mice received depleting anti-mouse CD4 or isotype control antibodies on d 14, 16, and 18 after termination of virulence inhibition. We used 800 µg per mouse, a dose reported to achieve efficient depletion 44 . Salmonella burdens were comparable in anti-CD4–treated mice vs . controls ( Fig 2d ), indicating limited impact of CD4 + T-cell. Thus, alternative mechanisms such as trained innate immunity may contribute 45 . This requires further investigations. Disease relapse but CFU control after enrofloxacin antibiotic To Compare anti-T3SS2 effect with clinically potent anti- Salmonella antibiotics. We treated infected mice with enrofloxacin, the veterinary prodrug of ciprofloxacin. Enrofloxacin cleared only ∼1 log CFU but also permitted control of bacterial loads after treatment albeit with relapsing disease symptoms ( Fig 2 e & f ). No synergy between T3SS-2 switch-off and antibiotic treatment It has been proposed that T3SS-2 activity is essential for intracellular Salmonella survival during antibiotic exposure 46 . Switching off T3SS-2 could thus sensitize Salmonella to antimicrobial chemotherapy. To test this, we treated infected mice from day 5 with the widely used fluoroquinolone ciprofloxacin using a dosing regimen as in human patients (10 mg/kg body weight, q12 h for 5 days) ( Fig 2g ). This treatment showed moderate efficacy (∼1 log reduction in splenic CFU), consistent with the limited reported efficacy against Salmonella in mice carrying functional SLC11A1 30 ( Fig. 2e – h ). Maintaining T3SS-2 in the OFF state throughout therapy did not further reduce bacterial loads or clinical signs. These findings align with prior work showing only additive, rather than synergistic, effects of an SsrB-targeting anti-virulence compound with colistin 22 . The results were also consistent with unaltered antibiotic survival of T3SS-2–deficient Salmonella 18 , 37 . Together, the data indicate that T3SS-2 activity is dispensable for Salmonella antibiotic tolerance even at advanced disease stages and that combining T3SS-2 inhibition with standard antibiotics is unlikely to yield synergy. Discussion Targeting virulence rather than directly killing bacteria is a promising strategy to control infection, offering more druggable targets, reduced selection for resistance, and less disruption of the microbiota 3 , 4 . However, anti-virulence compounds are not supposed to kill the bacteria directly 3 , 4 , posing a risk of bacterial persistence and relapsing disease after treatment termination. Such disarmed bacteria might be easier to clear by host immunity, enabling long-term infection control. Some anti-virulence compounds show impressive bacterial clearance in animal infection models 22 – 24 and partial effects in human patients 26 . Interpretation of such results remain challenging due to documented off-target effects in both bacteria and host cells. To assess the impact of specific targeting a single virulence system without perturbing bacterial or host physiology, we engineered a genetic system that enabled us to switch on and off virulence of Salmonella in infected mice using simple changes in the drinking water. We targeted T3SS-2 which is essential for Salmonella virulence 17 , 18 and exploited as target for two advanced anti-virulence compounds 22 , 26 . We inactivated the entire T3SS-2 by mutating the central transcription factor ssrB , and then complemented the strain with a DOX-inducible active ssrB allele. We showed that low DOX concentrations and without detectable effects in mice, fully restored virulence. Switching off T3SS-2 in infected mice arrested bacterial replication and – with some delay 47 – alleviated disease symptoms. Importantly, this transient, on-demand inhibition is more relevant to anti-virulence therapy compared to well-characterized permanently T3SS-2–deficient Salmonella mutants, which replicate poorly and cause no detectable disease symptoms and inflammation 18 , 37 . Switching off T3SS-2 for ten days did not clear infection, consistent with the long-term in vivo survival of T3SS-2 mutants and indicating that host immunity alone cannot eliminate disarmed Salmonella . These data also imply that prior reports of eradication by T3SS-2 inhibitors likely reflect off-target antibacterial or host effects 22 , 25 . Conversely, we did not observe escalating inflammation, suggesting that the anti-inflammatory functions of T3SS-2 – which might be important for shaping Salmonella microenvironments – exert limited effects at the organismal level. This supports the safety of T3SS-2 inhibition. When T3SS-2 was reactivated, Salmonella resumed replication, yet total bacterial loads remained stable, indicating that host immunity balanced Salmonella proliferation with killing. This differed markedly from the initial infection phase when active T3SS-2 drove exponential bacterial proliferation. Thus, transient T3SS-2 inhibition allowed the host to develop partially protective immunity. CD4 + T-cell depletion did not diminish this partial protection, suggesting alternative mechanisms that will require further investigation. Nevertheless, this partial control was insufficient to prevent relapse of disease symptoms. Comparison with clinically potent anti- Salmonella antibiotics revealed slight better, but again incomplete clearance and similar control of bacterial loads followed by relapse after treatment cessation. Combining T3SS-2 inhibition with antibiotics did not improve clearance. Together with prior in vivo data, these findings do not support a critical role for T3SS-2 in Salmonella survival during antibiotic exposure, contrary to proposals based on cell-culture studies 46 . T3SS-2 inhibition seemed as effective as standard antibiotics at suppressing Salmonella regrowth, suggesting that this approach could represent a suitable alternative. However, neither strategy prevented relapse in mice. Importantly, similar antibiotic regimens in human systemic salmonellosis achieve high cure rates and prevent relapse, indicating a limited predictive value of commonly mouse model. This could be related to stronger innate immunity / less virulent Salmonella strains in human patients compared to mouse infections. We used mice with restored SLC11A1 function which increases their anti- Salmonella resistance, but all infected animals would ultimately succumb, whereas human mortality is 10–30%, confirming lower clinical virulence. Taken together, these observations support prior concerns that standard mouse models are overly stringent for preclinical evaluation 48 and suggest that strains with better control of invasive Salmonella may more accurately predict the clinical utility of anti-virulence approaches 49 – 51 . In conclusion, we established a versatile model for transient on-target virulence inhibition in Salmonella . Selective inhibition of T3SS-2 did not eradicate infection, but it enabled post-treatment control, with efficacy approaching that of antibiotics that are highly effective against human systemic salmonellosis. Materials and Methods Bacterial strains and plasmids Salmonella strains used in this study were based on SME51, a prototrophic hisG Leu69 derivative of Salmonella enterica serovar Typhimurium SL1344 39 , 52 , 53 . Reporter strains carried inactivated ssrB by nonsense (STOP) mutations across the gene 54 (chromosomal coordinates in SL1344 GenBank FQ312003.1 : 1,433,019-1433657, M207X, M189X, G164X, E139X, M91X, M78X, G61X, M20X, M1X). Also, the strain carried low-copy episomal pSC101 derivatives with constitutive expression of mCherry , 55 or timer bac 39 from the P ybaJ promoter 54 (chromosomal coordinates in SL1344 GenBank FQ312003.1 : 528,632-528,081) and fusions of a doxycycline (DOX)-inducible promoter P tetA - ssrB driving expression of P sifB - GFP-ova coding for a degradable variant of the green fluorescent protein GFP.mut2 56 . All plasmids were constructed using Gibson assembly 57 using primers listed in Table S1 and S2 . View this table: View inline View popup Download powerpoint Table 1S. View this table: View inline View popup Download powerpoint Table 2S. In-vitro P tetA -ssrB induction Bacteria were grown overnight in MES-MM (100 mM MES-KOH pH 5.5, 0.4% glycerol, 15 mM NH 4 Cl, 1.5 mM K 2 SO 4 , 3 mM KH 2 PO 4 ) containing 90 mg/L streptomycin and 50 mg/L kanamycin 30 . Day cultures were inoculated at an OD 600 nm of 0.05 and grown for 2 h. Doxycycline (0.125 mg/L) was added to induce P tetA -ssrB . Cultures were incubated for 4 h at 37 °C with shaking (180 rpm), after which samples were withdrawn, diluted 1:10 in PBS and fluorescence intensity was measured. Mice All animal experiments were approved (license 2239, Kantonales Veterinäramt Basel) and performed according to local guidelines (Tierschutz-Verordnung, Basel) and the Swiss animal protection law (Tierschutz-Gesetz). Mice (C57BL/6J Slc11A1 r/r 34 ) were housed at 22°C (-2°C/+3°C), relative humidity of 55 +/-10%, and a 12h/12h dark/light cycle. We infected mice at 10 to 15 weeks of age and used both female and male mice which show comparable results for Salmonella infections 34 . We estimated sample size by a sequential statistical design. We first infected two to three mice based on effect sizes and variation observed in our previous studies 34 , 58 and used the results to estimate group sizes for obtaining statistical significance with sufficient power. Mice were exposed to DOX and 4% sucrose one day pre-infection in their drinking water. Mice were then infected by tail-vein injection of ∼1,200 CFU Salmonella grown to late-log phase in Lennox LB containing 10 mM MgCl 2 (which increased consistency across experiments). The inoculum size was determined by plating. Intravenously infected mice show similar Salmonella growth rates 39 and Salmonella localization in spleen 59 compared to orally infected mice but exhibit less variation in Salmonella tissue loads between individual mice, and thus require fewer experimental animals for detecting differences with the same statistical power. For plating, mice were euthanized with carbon dioxide and spleen was homogenized in PBS containing 0.2% Triton X-100. Salmonella load was determined by plating on lysogeny broth agar. Activity of P sifB - gfp-ova and Salmonella survival were determined by comparing flow cytometry data and corresponding CFU counts of Salmonella populations. Mice were scored daily for disease signs (physical appearance: normal – 0, lack of grooming – 1, piloerection and nasal ocular discharge – 2, hunched up – 3; behavior (mobile – 0, reduced mobility – 2, immobile – 3). Antibodies For depletion, purified anti-mouse CD4 (clone GK1.5) or isotype control (rat IgG2b) antibodies (obtained from Bio X Cell), a dose of 800 μg per mouse and administered intraperitoneally on days 14, 16, and 18 post-infection. Flow cytometry The spleen was homogenized in 6 mL ice-cold phosphate-buffered saline containing 0.2% Triton X-100. All samples were kept on ice until and during analysis. Large host cell fragments were removed by repeated centrifugations at 100xg for 10 min at 4°C. Relevant spectral parameters were recorded in a FACS Fortessa II (Becton, Dickinson) with ‘low’ speed, using a threshold on sideward scatter (SSC) to exclude electronic noise. We used the following channels: GFP and green TIMER bac component, excitation 488 nm, emission 502-525 nm (“green”); mCherry and red TIMER bac component, excitation 561 nm, emission 595-664 nm (“red”) (with 633 nm laser switched off); yellow and orange autofluorescence, excitation 488 nm, emission 533-551 nm, 573-613 nm; orange autofluorescence, excitation 445 nm, emission 573-613 nm. All parameters were measured as ‘height’. Data were processed with FlowJo 10.6.1 and MATLAB R2020b. TIMER bac fluorescence log color-ratios were converted into Salmonella division rates based on previously established calibration data 39 . Quantification and statistical analysis Statistical tests were performed with GraphPad Prism 9.3.1 as indicated in the figure legends. The respective comparisons are denoted in the Figure panels. We log-transformed CFU data to approximate normal distributions 60 . Comparison of two groups were done by t-test. Matched measurements from the same infected mouse (such as subsets with differential promoter activity, or mutant and wild-type Salmonella loads) were analyzed by paired tests. Comparisons of three or more groups were done by one-way ANOVA. The impact of two factors was analyzed by two-way ANOVA. All tests were two-tailed. We deliberately avoided specifying a particular significance threshold for P -values ( e . g ., α = 0.05, 0.01, or 0.001) to avoid excessive emphasis on arbitrary cut-offs 61 . Funding This research was supported by Swiss National Science Foundation (10000546) and AntiResist (grant 180541) to DB. Author contributions Outlined the study: DB and AA, performed experiments: AA, BC, SN, HA analyzed data: AA and DB, interpreted data: DB and AA, wrote manuscript: DB and AA. Competing interests Authors declare no competing interests. Data and materials availability All data is available in the manuscript or the supplementary materials. 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Share Long-term control of Salmonella after transient T3SS-2 inhibition Alaa Alhayek , Beatrice Claudi , Sila Nizam , Helen Atassi , Dirk Bumann bioRxiv 2025.10.30.685576; doi: https://doi.org/10.1101/2025.10.30.685576 Share This Article: Copy Citation Tools Long-term control of Salmonella after transient T3SS-2 inhibition Alaa Alhayek , Beatrice Claudi , Sila Nizam , Helen Atassi , Dirk Bumann bioRxiv 2025.10.30.685576; doi: https://doi.org/10.1101/2025.10.30.685576 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7633) Biochemistry (17680) Bioengineering (13888) Bioinformatics (41927) Biophysics (21445) Cancer Biology (18585) Cell Biology (25491) Clinical Trials (138) Developmental Biology (13373) Ecology (19897) Epidemiology (2067) Evolutionary Biology (24308) Genetics (15606) Genomics (22494) Immunology (17736) Microbiology (40385) Molecular Biology (17175) Neuroscience (88583) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4822) Physiology (7641) Plant Biology (15149) Scientific Communication and Education (2045) Synthetic Biology (4293) Systems Biology (9822) Zoology (2271)