{"paper_id":"1911900a-5e0b-4611-b6ca-c5626a91a5c8","body_text":"1 \n \nPhenotypic Characterization of Signature-Tagged Mutants Identifies 1 \nPhysiological Determinants of Vibrio vulnificus  Fitness 2 \n 3 \nKohei Yamazaki, a Takehiro Kado, a,b Takashige Kashimoto, a# 4 \n 5 \naLaboratory of Veterinary Public Health, School of Veterinary Medicine, Kitasato 6 \nUniversity, Aomori, Japan  7 \nbDepartment of Biology, Missouri State University, 901 South National Avenue, 8 \nSpringfield, MO 65897  9 \n 10 \nRunning Title; In vivo fitness of Vibrio vulnific us 11 \n 12 \n#Address correspondence to  Takashige Kashimoto, 13 \nkashimot@vmas.kitasato -u.ac.jp   14 \nKohei Yamazaki and Takehiro Kado contributed equally to this work. Author order 15 \nwas determined in order of increasing seniority.  16 \nWord count: 221 words (abstract),  3127  words (Introduction through Discussion)  17 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n2 \n \nAbstract  18 \nVibrio vulnificus  is an opportunistic marine pathogen that causes severe 19 \nwound-associated and systemic infections. Following entry into the host, the 20 \nbacterium must rapidly adapt to host-associated stresses that differ substantially 21 \nfrom those encountered in aquatic environments. However, the physiological 22 \nfunctions supporting bacterial fitness during infection remain incompletely 23 \nunderstood. Previously, we applied signature-tagged mutagenesis (STM) to 24 \nidentify genes required for  V. vulnificus  survival during host infection, revealing 25 \nnumerous loci that did not correspond to classical toxin-encoding genes. In the 26 \npresent study, we extended this genome-wide screen by linking STM-identified 27 \nmutations to observable fitness-related phenotypes. Functional annotation revealed 28 \nenrichment of genes associated with chemotaxis, flagellar motility, regulation, 29 \nmetabolism, and poorly characterized functions. Phenotypic analyses showed that 30 \nmany STM-derived mutants exhibited defects in swimming motility and altered 31 \ncolony surface properties. Bioluminescence imaging further revealed distinct 32 \npatterns of impaired persistence and dissemination within host tissues, while 33 \nseveral mutants displayed increased susceptibility to phagocytic stress in an 34 \nHL-60-derived neutrophil model. Notably, some regulatory mutants affecting 35 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n3 \n \nglobal signaling pathways exhibited impaired tissue dissemination despite 36 \nretaining resistance to phagocytic stress, indicating the presence of fitness 37 \ndeterminants that operate independently of classical surface-associated or 38 \ncytotoxic traits. Together, these findings demonstrate that V. vulnificus  fitness 39 \nduring infection depends on diverse physiological pathways beyond classical 40 \nvirulence factors, highlighting the value of phenotype-centered analyses for 41 \nunderstanding bacterial adaptation in host-associated environments. 42 \nImportance 43 \nVibrio vulnificus  causes rapidly progressive wound infections and septicemia, yet 44 \nthe bacterial functions that support fitness within host environments remain 45 \nincompletely defined. While substantial effort has focused on canonical virulence 46 \nfactors and regulation, increasing evidence suggests that successful infection also 47 \ndepends on broader physiological adaptation. In this study, we link 48 \nsignature-tagged mutagenesis with systematic phenotypic analyses to define 49 \nphysiological determinants of  V. vulnificus  fitness during host-associated infection. 50 \nOur results demonstrate that genes involved in motility, regulatory signaling, 51 \nmetabolism, and stress tolerance collectively shape bacterial persistence and 52 \ndissemination in host tissues. Notably, we identify regulatory and metabolic 53 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n4 \n \ndeterminants that influence fitness independently of classical surface-associated or 54 \ncytotoxic traits, highlighting noncanonical pathways that contribute to pathogenic 55 \nsuccess. By integrating genome-wide screening with phenotype-centered analyses, 56 \nthis work advances understanding of how physiological adaptation underpins V. 57 \nvulnificus  infection and provides a framework for studying bacterial fitness 58 \nalongside established concepts of bacterial virulence. 59 \nKeywords  60 \nVibrio vulnificus ; host-associated environments ; in vivo  fitness; signature-tagged 61 \nmutagenesis (STM); motility; two-component signaling; amino-sugar metabolism; 62 \nenvironmental adaptation; bioluminescence imaging 63 \nIntroduction  64 \nThe public health relevance of Vibrio vulnificus  has increased markedly in recent 65 \nyears. Rising sea surface temperatures associated with global climate change have 66 \nexpanded the geographical range of this marine bacterium, leading to increased 67 \ncase numbers and reports of severe wound-associated infections in regions that 68 \nwere previously unaffected (1 , 2). Because V. vulnificus  infection is frequently 69 \nassociated with rapidly progressive necrotizing soft tissue infections and high 70 \nmortality, particularly following wound exposure, it represents a growing public 71 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n5 \n \nhealth concern worldwide (3 –8). As opportunities for environmental exposure 72 \ncontinue to grow, there is an increasing need to understand how V. vulnificus  73 \nadapts to host-associated environments following entry into the human body. 74 \nVibrio vulnificus  is a marine bacterium that opportunistically invades host tissues 75 \nthrough wounds, where it encounters physicochemical constraints, nutrient 76 \nlimitation, and host-derived biotic pressures that differ substantially from those in 77 \nexternal aquatic environments. Successful proliferation in soft tissues is known to 78 \ndepend on several bacterial traits that support survival under these stresses. The 79 \nRTX toxin has been widely reported to exert cytotoxic effects on host immune 80 \ncells, particularly neutrophils, thereby impairing early innate immune defenses (9, 81 \n10). In addition, the capsular polysaccharide contributes to resistance against 82 \nphagocytosis, promoting bacterial persistence in host environments (11). 83 \nChemotaxis and flagellar motility further enable bacterial migration and spatial 84 \nexpansion within infected tissues, facilitating access to niches permissive for 85 \ngrowth ( 12). 86 \nAlthough these traits are established contributors to pathogenicity, they represent 87 \nonly a subset of the bacterial functions required for survival in complex 88 \nhost-associated environments. Increasing evidence from genome-wide analyses 89 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n6 \n \nindicates that many genes essential for in vivo  fitness do not correspond to 90 \nclassical virulence determinants but instead encode physiological functions 91 \ninvolved in regulation, motility, and metabolism ( 13-16). These findings suggest 92 \nthat environmental adaptation, rather than toxin-mediated damage alone, plays a 93 \ncentral role during early stages of infection. 94 \nPreviously, we applied signature-tagged mutagenesis (STM) to identify genes 95 \nrequired for V. vulnificus  fitness during wound infection (13). That study 96 \ngenerated a genome-wide map of candidate loci contributing to survival within 97 \nhost tissues and revealed that many STM-identified genes were not canonical 98 \nvirulence factors. Instead, a large proportion of the recovered loci encoded 99 \ncomponents of motility systems, regulatory elements, metabolic enzymes, or 100 \nproteins of unknown function, supporting the idea that physiological adaptation is 101 \ncritical for persistence in vivo.  102 \nIn the present study, we extend this work by focusing on phenotypic 103 \ncharacterization of representative STM-identified mutants. To directly link STM 104 \nselection with observable fitness-related traits, we employed  in vivo  105 \nbioluminescence imaging to monitor bacterial persistence and dissemination in 106 \nreal time, together with assays measuring tolerance to phagocyte-associated stress. 107 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n7 \n \nThis approach allows evaluation of how specific physiological pathways 108 \ncontribute to environmental fitness in host tissues without addressing the 109 \nmolecular mechanisms underlying individual virulence factors. 110 \nUsing this strategy, we show that mutants defective in motility ( motX ), regulatory 111 \nsignaling ( barA  and luxO ), and amino-sugar metabolism ( gpsK ) exhibit reduced in 112 \nvivo  fitness and diminished tolerance to phagocytic stress. Together, these 113 \nfindings update and extend our previous STM analysis by integrating functional 114 \nphenotyping and real-time imaging, providing insight into the physiological 115 \nsystems that support V. vulnificus  adaptation to host-associated environments. 116 \nMethods  117 \nAnimals and Ethics Statement  118 \nFive -week -old female C57BL/6 or BALB/c mice (Charles River Laboratories 119 \nJapan) were housed under SPF conditions with ad libitum access to food and water, 120 \nfollowing a 12:12 h light -dark cycle. All animal experiments were approved by the 121 \nInstitutional Animal Care and Use Committee of Kitasato University, in 122 \naccordance with JALAS guidelines, consistent with our previous reports  123 \n(Approval No. 19-218). 124 \nBacterial Strains  125 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n8 \n \nVibrio vulnificus  CMCP6 (clinical isolate) was used as the wild-type (WT) strain. 126 \nTransposon mutants were generated as described previously (13). Briefly, 127 \nEscherichia coli  BW19795 carrying the signature-tagged mini-Tn5Km2 128 \ntransposon on the suicide vector pUT was used as the donor strain for conjugation. 129 \nTransposons were introduced into Vibrio vulnificus  by biparental mating, and 130 \ntransconjugants were selected on LB agar plates containing kanamycin under 131 \nappropriate conditions. For genetic complementation, the target gene was 132 \namplified by PCR and cloned into the low-copy-number plasmid pACYC184 using 133 \nIn-Fusion cloning reactions (Clontech, TaKaRa, Shiga, Japan), according to th e 134 \nmanufacturer’s instructions. The resulting complementing plasmid was introduced 135 \ninto V. vulnificus  by electroporation. Transformants were selected on LB agar 136 \nplates containing chloramphenicol (10 µg/ml) and incubated overnight at 37°C. 137 \nAll strains were grown aerobically in Luria –Bertani (LB) broth or on LB agar at 138 \n37 °C, as described previously. When appropriate, antibiotics were added at the 139 \nfollowing concentrations: rifampicin 50 µg/ml, kanamycin 100 µg/ml, or 140 \nampicillin 100 µg/ml.  141 \nGrowth and Inoculati on Conditions  142 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n9 \n \nOvernight cultures were diluted 1:20 into fresh LB medium and incubated for 2 h 143 \nat 37°C with agitation (163 rpm). Bacterial cells were harvested, washed with PBS 144 \ncontaining 0.1% gelatin, and resuspended to the desired concentration. Mice wer e 145 \ninoculated subcutaneously in the caudal thigh with 1 × 10⁶ CFU of wild -type or 146 \nmutant strains. Infected mice were monitored at defined time points (3 –12 h 147 \npostinfection) for clinical signs.  148 \nIdentification of transposon insertion sites  149 \nThe transposon insertion sites were determined using arbitrarily primed PCR as 150 \ndescribed previously (13). Briefly, genomic DNA was isolated from each 151 \ntransposon mutant, and PCR was performed using primers specific to the 152 \ntransposon together with arbitrary primers targeting the Vibrio vulnificus  genome. 153 \nThe resulting PCR products were sequenced, and insertion sites were identified by 154 \nsequence homology searches against the V. vulnificus  genome database.  155 \nMotility  assay  156 \nSwimming motility was assessed on 0.3% ag ar, representing a low -viscosity 157 \nenvironment relevant to tissue and fluid interfaces  (12,13). 158 \nCapsule Opacity Assay (CPS phenotype)  159 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n10 \n \nOvernight cultures were spotted onto LB agar and incubated 12 h at 37 °C. Colony 160 \ntranslucency/opacity was visually inspected  (11,13). 161 \nIn vivo  bioluminescence imaging  162 \nTo evaluate persistence and dissemination, WT and mutants were transformed with 163 \npXen -13 ( luxCDABE ). Bioluminescent WT and mutant strains were visualized 164 \nusing an IVIS -200 imaging system (PerkinElmer). Mice were ane sthetized with 165 \nisoflurane, and images were acquired with a fixed exposure time of 1 minute at 3, 166 \n6, 9, and 12 hours after subcutaneous infection  (15, 17).  167 \nOpsonophagocytic survival assay  168 \nNeutrophil -like cells were generated by differentiating HL -60 cells with 1.25% 169 \ndimethyl sulfoxide (DMSO) for 5 days , as described previously  (16) . Bacterial 170 \ncells were incubated with differentiated HL -60 cells at a multiplicity of infection 171 \n(MOI) of 1:20 in the presence of 10% human serum for 45 min at 37°C with gentle 172 \nrotation. Following incubation, host cells were lysed by treatment with 0.05% 173 \nsaponin, and samples were serially diluted and plated onto LB agar for 174 \nenumeration of surviving CFU.  Bacterial survival was calculated by comparing 175 \nCFU recovered after incubation wi th HL -60 cells to CFU recovered from control 176 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n11 \n \nsamples incubated for the same duration under identical conditions in the absence 177 \nof HL -60 cells.  178 \nStatistical analysis  179 \nAll statistical analyses were performed using GraphPad Prism (version 8). 180 \nQuantitative comparisons of bacterial CFU counts were evaluated using the Mann –181 \nWhitney U test, as the data did not follow a normal distribution. For all 182 \nexperiments, p values of <  0.05 were considered statistically significant.  183 \nResults  184 \nFunctional catego rization of STM -identified genes   185 \nTo evaluate the functional characteristics of genes identified by the STM screen, 186 \nwe first examined the distribution of annotated functions among the selected loci. 187 \nAll STM -derived mutants exhibited growth kinetics comparable to WT under 188 \nstandard in vitro culture conditions (data not shown), indicating that the observed 189 \nattenuation was not attributable to intrinsic growth defects.  The STM -attenuated 190 \ngene set was enriched for genes associated with chemotaxis and flage llar motility, 191 \nincluding multiple components of the basal body, motor complex, hook, and 192 \nchemotactic signaling pathways  (Table 1) . In addition to motility -associated genes, 193 \nthe STM -identified loci included regulatory elements, metabolic enzymes, stress 194 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n12 \n \nresponse proteins, and factors involved in chromosome maintenance and cell 195 \ndivision  (Table 1) . A substantial fraction of the identified genes encoded proteins 196 \nannotated as hypothetical or with limited functional characterization.  197 \nMotility defects among STM -identified mutants  198 \nTo assess whether STM -identified mutations were associated with defects in 199 \nmotility -related phenotypes, all mutants listed in Table 1 were subjected to a 200 \nswimming motility assay as an initial phenotypic screen. WT exhibited robust 201 \nradial e xpansion on soft -agar plates, whereas multiple STM -derived mutants 202 \ndisplayed marked reductions in motility (Fig. 1).  203 \nAs expected, disruptions in genes encoding flagellar structural components and 204 \nmotor -associated proteins , including motX, fliH, fliM, fliF , fliK, flgE, flgH, flgI,  205 \nand flhF , resulted in severe motility defects  (Fig. 1). Mutations in chemotactic 206 \nsignaling proteins ( cheY  and cheW ) similarly impaired swimming behavior, 207 \nconsistent with their established roles in directed bacterial movement.  In a ddition 208 \nto these canonical motility -related genes, several mutants carrying insertions in 209 \ngenes not directly annotated as motility components also exhibited reproducible 210 \nreductions in swimming motility. These included genes encoding a siderophore 211 \nreceptor (iutA ), predicted membrane proteins, lipase -related proteins, exported 212 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n13 \n \nproteins, chromosome segregation factors ( parA ), the sigma -54 transcription 213 \nfactor ( rpoN ), and an N -end rule protein modification enzyme ( aat) (Fig. 1). These 214 \nfindings indicate that div erse physiological and regulatory functions contribute 215 \nindirectly to motility -associated behaviors in V. vulnificus . 216 \nCapsule -associated colony opacity variations  217 \nColony morphology was assessed as an indicator of surface -associated properties 218 \npotentially relevant to host adaptation. WT formed opaque colonies on LB agar, 219 \nwhereas several STM -derived mutants produced colonies with increased 220 \ntranslucency (Fig. 2). Notab ly, mutants carrying insertions in the regulatory 221 \nsignaling gene barA  and a gene encoding a glycosyltransferase exhibited a 222 \ntranslucent colony phenotype comparable to that observed in the environmental 223 \nisolate control strain E4  (Fig. 2) . This altered colon y morphology was 224 \nreproducibly observed in mutants affecting regulatory signaling pathways and 225 \nglycosylation -related functions. In contrast, STM -derived mutants disrupted in 226 \nflagellar motility or chemotactic signaling genes retained colony opacity 227 \ncomparabl e to that of the wild -type strain, indicating that the observed 228 \ntranslucency phenotype was not a general consequence of STM mutagenesis but 229 \nwas associated with specific functional categories.  230 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n14 \n \nReduced persistence and dissemination in soft tissues among repr esentative 231 \nSTM-derived mutants  232 \nTo determine whether STM -identified mutations impaired bacterial fitness in soft 233 \ntissues, bioluminescent derivatives of selected mutants were monitored during 234 \nsubcutaneous infection using in vivo  bioluminescence imaging (IVIS). WT 235 \nexhibited progressive dissemination of luminescent signals from the inoculation 236 \nsite, followed by signal attenuation by 12 h postinfection, consistent with active 237 \npersistence, expansion, and subsequent invasion into deep er soft tissues (Fig. 3) 238 \n(12, 17). 239 \nBased on IVIS signal dynamics, STM -derived mutants could be broadly classified 240 \ninto three phenotypic groups. The first group displayed behavior in soft tissues 241 \ncomparable to that of WT. Mutants carrying insertions in VV1_ 0388 and 242 \nVV1_0778 showed sustained luminescent signals and spatial dissemination similar 243 \nto those observed for WT, indicating that disruption of these loci did not markedly 244 \nimpair persistence or spread in soft tissues.  245 \nThe second group exhibited severe def ects in dissemination from the infection site. 246 \nAs expected, the motility -deficient mutant motX::Tn  failed to spread within soft 247 \ntissues, consistent with previous observations that bacterial motility is essential 248 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n15 \n \nfor expansion and dissemination in this infe ction model (12). Similarly, mutants 249 \ndisrupted in regulatory signaling pathways ( luxO::Tn  and barA::Tn ) showed 250 \nrestricted luminescent signals that remained localized near the inoculation site, 251 \nresembling the phenotype of the motility -deficient mutant ( 13). 252 \nThe third group showed limited dissemination accompanied by prolonged 253 \npersistence of luminescent signals at the infection site. The chemotaxis -deficient 254 \nmutant cheW::Tn  exhibited spread within soft tissues; however, luminescent 255 \nsignals remained detectable  over the observation period. This phenotype is 256 \nconsistent with previously described behavior of chemotaxis mutants that retain 257 \nmotility but exhibit impaired invasion into deeper tissue compartments (12). A 258 \nsimilar IVIS phenotype was observed for several a dditional STM -derived mutants, 259 \nincluding mukB ::Tn , ftsX ::Tn , gpsK ::Tn , VV1_0055::Tn, VV1_0056::Tn, 260 \nVV2_0147::Tn, VV2_0122 ::Tn , VV1_0501::Tn, and VV2_0146::Tn.  261 \nCollectively, IVIS analysis revealed distinct phenotypic classes among 262 \nSTM-derived mutants in sof t tissues, reflecting differential defects in bacterial 263 \npersistence, dissemination within soft tissues, and invasion into deeper tissue 264 \ncompartments.  265 \nIncreased susceptibility of STM -derived mutants to phagocytic stress  266 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n16 \n \nBecause early host responses to subcutaneous infection involve rapid recruitment 267 \nof phagocytic cells, STM -derived mutants were examined for tolerance to 268 \nphagocytic stress using differentiated HL -60 cells as a neutrophil -like cell  model. 269 \nWT demonstrated sub stantial survival following exposure to phagocytic cells (Fig. 270 \n4). The barA ::Tn mutant exhibited survival comparable to that of WT (Fig. 4), 271 \nindicating that disruption of barA  did not markedly impair resistance to phagocytic 272 \nstress under these conditions.  273 \nIn contrast, several STM -derived mutants showed significantly reduced survival 274 \nafter incubation with HL -60 cells. Mutants carrying insertions in motX , luxO , 275 \nVV1_0388, and gpsK  displayed pronounced decreases in survival relative to WT 276 \n(Fig. 4), indicating i ncreased susceptibility to phagocyte -associated stress. These 277 \nreductions were consistently observed across independent experiments.  278 \nTo confirm that the observed phenotypes were attributable to the respective 279 \ntransposon insertions, complementation analyses were performed. Introduction of 280 \nthe corresponding wild -type genes restored survival to levels comparable to WT, 281 \nconfirming that the increased susceptibility to phagocytic stress resulted from 282 \ndisruption of the targeted loci.  283 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n17 \n \nDiscussion  284 \nIn this study, we extended our previous STM -based analysis by integrating 285 \ntargeted phenotypic characterization to define physiological traits associated with 286 \nVibrio vulnificus  fitness in host -associated environments. By combining STM 287 \nselection with motilit y assays, evaluation of surface -associated properties, 288 \nbioluminescence -based imaging in soft tissues, and phagocytic stress assays, we 289 \ndirectly linked STM -identified loci to observable fitness -related phenotypes 290 \nwithout addressing the molecular mechanisms underlying individual virulence 291 \nfactors.  292 \nThe functional composition of STM -identified genes provides initial validation of 293 \nthe screening strategy. The enrichment of chemotaxis - and flagellar motility –294 \nassociated genes among STM -attenuated mutants is consist ent with the established 295 \nimportance of directed movement for bacterial expansion within soft tissues  (Table 296 \n1) (12, 13). Motility enables bacteria to explore heterogeneous host environments 297 \nand access niches permissive for growth, and its repeated recovery  in STM -based 298 \nscreens supports the view that motility -related functions represent core 299 \nphysiological requirements for fitness during early infection (12).  However, the 300 \nmotility -deficient motX  mutant retained substantial resistance to phagocytic stress 301 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n18 \n \n(Fig . 4). In the HL -60 survival assay, motX ::Tn exhibited a median survival of 302 \n83.5%, indicating that the absence of motility alone does not render V. vulnificus  303 \ncompletely susceptible to phagocytic killing. This residual resistance is likely 304 \nsupported by addi tional virulence -associated traits retained by the motX  mutant, 305 \nsuch as capsular polysaccharide and RTX toxin production  (9,10,12 ). 306 \nIn contrast, genes encoding the RTX toxin were not recovered among 307 \nSTM-selected loci  (Table 1) . This absence likely reflects  a methodological 308 \ncharacteristic of STM -based negative selection screens rather than a lack of 309 \ncontribution of RTX to virulence. Because STM involves pooled infection with 310 \nmultiple mutant strains, secreted virulence factors such as toxins can be 311 \nfunctional ly complemented by neighboring bacteria. Under these conditions, 312 \nmutants defective in toxin production may not exhibit a competitive disadvantage 313 \nand therefore escape negative selection. Consequently, STM preferentially 314 \nidentifies cell -autonomous physiolog ical functions required for survival and 315 \npersistence, while diffusible virulence factors are underrepresented  (13,14 ). 316 \nBeyond motility -associated genes, the STM -identified set included regulatory 317 \nelements, metabolic enzymes, stress response proteins, and f actors involved in 318 \nchromosome maintenance and cell division  (15,16,18-23). Notably, a substantial 319 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n19 \n \nfraction of the recovered loci encoded proteins of unknown or poorly 320 \ncharacterized function. The recurrence of such genes among STM -attenuated 321 \nmutants suggests that V. vulnificus  relies on additional, incompletely understood 322 \nphysiological processes to adapt to host -associated environments. These findings 323 \nunderscore the value of genome -wide screening approaches for uncovering fitness 324 \ndeterminants that are not readily predicted from existing virulence models.  325 \nPhenotypic analyses revealed that disruptions in motility and chemotaxis strongly 326 \nimpair bacterial migration  (Fig.  1), confirming tha t these traits are tightly linked to 327 \nfitness in soft tissues  (Fig. 3). Although luxO  and barA  mutants retained normal 328 \nswimming motility in vitro (Fig. 1), both mutants exhibited severe defects in 329 \npersistence and dissemination within soft tissues (Fig. 3), resembling the 330 \nphenotype of the motility -deficient motX  mutant. This discrepancy indicates that 331 \nregulatory pathways controlled by LuxO and BarA  contribute to fitness in soft 332 \ntissues through mechanisms that are independent of flagellar motility. Rather than 333 \naffecting bacterial movement per se, these regulators likely coordinate additional 334 \nphysiological processes required for growth, persistence, o r spatial expansion 335 \nwithin host tissues.  336 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n20 \n \nAlterations in colony opacity among selected STM mutants further suggest that 337 \nregulatory and metabolic pathways influence bacterial surface architecture. The 338 \nglycosyltransferase mutant (VV1_0778::Tn) formed transluc ent colonies, which 339 \nsuggests reduced capsular polysaccharide production (Fig. 2), yet it retained the 340 \nability to disseminate within soft tissues as efficiently as WT (Fig. 3). In contrast, 341 \nthe barA ::Tn mutant also exhibited a translucent colony phenotype ( Fig. 2), but 342 \nIVIS analysis revealed marked defects in tissue dissemination (Fig. 3), despite this 343 \nmutant retaining resistance to phagocytic stress (Fig. 4). These observations 344 \nindicate that neither colony opacity nor resistance to phagocytic stress alone c an 345 \nreliably predict bacterial fitness in soft tissues, suggesting that additional factors 346 \nindependent of capsule production and motility contribute to successful growth 347 \nand spread in this environment.  348 \nThe IVIS analysis further revealed distinct phenotypic classes among 349 \nSTM-derived mutants, reflecting differential defects in persistence, dissemination, 350 \nand invasion into deeper tissue compartments  (Fig. 3) (12) . In particular, 351 \nregulatory mutants ( luxO ::Tn and barA ::Tn) exhibited dissemination defects 352 \ncomparab le to those of motility -deficient strains, despite differing phenotypes in 353 \nother assays. LuxO is a central regulator of quorum -sensing pathways in V. 354 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n21 \n \nvulnificus  and has been implicated in the control of virulence -associated gene 355 \nexpression, including facto rs linked to RTX toxin regulation ( 18-21). Although 356 \nRTX genes were not directly identified in the STM screen, impaired regulation of 357 \ntoxin expression or secretion may contribute indirectly to the reduced fitness of 358 \nluxO  mutants in soft tissues.  359 \nConsistent with the IVIS analysis, several STM -derived mutants displayed 360 \nincreased susceptibility to phagocytic stress in an HL -60-derived neutrophil model 361 \n(Fig. 4). Reduced survival among motility -, regulatory -, and metabolism -defective 362 \nmutants indicates that these pathways contribute to tolerance of host -derived 363 \nbiotic pressures encountered during early infection. Notably, barA ::Tn retained 364 \nresistance to phagocytic stress despite exhibiting impaired dissemination in soft 365 \ntissues, further supporting the notion that f itness in this environment reflects the 366 \nintegration of multiple physiological traits rather than reliance on a single immune 367 \nevasion mechanism.  368 \nGpsK is a glucosamine -specific kinase that catalyzes the conversion of 369 \nglucosamine to glucosamine -6-phosphate, r epresenting the initial step of amino 370 \nsugar utilization in Vibrio  species ( 23). This pathway constitutes a central 371 \ncomponent of chitin -derived nutrient metabolism and has been implicated in 372 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n22 \n \nenvironmental adaptation of marine Vibrios  (24). Although disrupti on of gpsK  did 373 \nnot result in detectable growth impairment under in vitro  conditions, loss of this 374 \nenzyme may restrict metabolic flexibility in host -associated environments, where 375 \naccessible carbon and nitrogen sources are limited. Such constraints could 376 \ncompromise bacterial fitness in soft tissues, contributing to the delayed tissue 377 \ninvasion and increased susceptibility to phagocytic stress observed for the 378 \ngpsK ::Tn mutant. These findings suggest that gpsK  supports in vivo fitness not by 379 \nenhancing basal gro wth capacity but by enabling metabolic adaptation to 380 \nnutrient -limited and stress -rich host environments.  381 \nTaken together, the results of this study indicate that Vibrio vulnificus  fitness in 382 \nhost -associated environments is governed by a network of physiolog ical functions 383 \nencompassing motility, regulatory signaling, metabolism, and stress tolerance. 384 \nMany of the identified determinants do not correspond to classical virulence 385 \nfactors but instead contribute to bacterial adaptation under host -imposed 386 \nconstraints . By linking STM -based selection with phenotypic outcomes, this work 387 \nprovides further insight into how environmental adaptation underpins bacterial 388 \npersistence during early stages of infection and highlights the value of 389 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n23 \n \nphenotype -centered approaches for d issecting in vivo fitness beyond canonical 390 \nvirulence paradigms.  391 \nReference  392 \n1. Baker-Austin C, Oliver JD, Alam M, et al. 2018. Vibrio  spp. infections. Nat 393 \nRev Dis Primers  4:8. https://doi.org/10.1038/s41572-018-0005-8 394 \n2. Baker-Austin C, Trinanes JA, Taylor NGH, et al. 2013. Emerging Vibrio  risk 395 \nat high latitudes in response to ocean warming. Nat Clim Chang  3:73 –77. 396 \nhttps://doi.org/10.1038/nclimate1628 397 \n3. Stevens DL, Bryant AE. 2017. Necrotizing soft-tissue infections. N Engl J 398 \nMed 377:2253 –2265. https://doi.org/10.1056/NEJMra1600673 399 \n4. Skrede S, Bruun T, Rath E, Oppegaard O. 2020. Microbiological etiology of 400 \nnecrotizing soft tissue infections. Adv Exp Med Biol  1294:53 –71. 401 \nhttps://doi.org/10.1007/978-3- 030-57616-5_5 402 \n5. Peetermans M, de Prost N, Eckmann C, et al. 2020. Necrotizing skin and 403 \nsoft-tissue infections in the intensive care unit. Clin Microbiol Infect  26:902 –404 \n909. https://doi.org/10.1016/j.cmi.2020.02.005 405 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n24 \n \n6. Diaz JH. 2014. Skin and soft tissue infections following marine injuries and 406 \nexposures in travelers. J Travel Med  21:207 –213. 407 \nhttps://doi.org/10.1111/jtm.12115 408 \n7. Finkelstein R, Oren I. 2011. Soft tissue infections caused by marine bacterial 409 \npathogens: epidemiology, diagnosis, and management. Curr Infect Dis Rep  410 \n13:470 –477. https://doi.org/10.1007/s11908-011-0199-3 411 \n8. Oliver JD. 2005. Wound infections caused by Vibrio vulnificus  and other 412 \nmarine bacteria. Epidemiol Infect  133:383 –391. 413 \nhttps://doi.org/10.1017/S0950268805003894 414 \n9.  Lo HR, Lin JH, Chen YH, Chen CL, Shao CP, Hor LI. 2011. RTX toxin 415 \nenhances the su rvival of Vibrio vulnificus  during infection by protecting the 416 \norganism from phagocytosis. J Infect Dis  203:1866 –1874. 417 \nhttps://doi.org/10.1093/infdis/jir122  418 \n10. Kim YR, Lee SE, Hyun K, Yeom JA, Na HS, Kim SY, et al. 2008. Vibrio 419 \nvulnificus  RTX toxin kills host cells only after contact of the bacteria with 420 \nhost cells. Cell Microbiol  10:848 –862. 421 \nhttps://doi.org/10.1111/j.1462-5822.2007.01088.x 422 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n25 \n \n11. Pettis GS, Mukerji AS. 2020. Structure, function, and regulation of the 423 \nessential virulence factor capsular polysaccharide of Vibrio vulnificus . Int J 424 \nMol Sci  21:3259. https://doi.org/10.3390/ijms21093259 425 \n12. Yamazaki K, Kashimoto T, Kado T, Akeda Y, Yoshioka K, Kodama T. 2020. 426 \nChemotactic invasion in deep soft tissue by Vibrio vulnificus  is essential for 427 \nthe progression of necrotic lesions. Virulence  11:839 –847. 428 \nhttps://doi.org/10.1080/21505594.2020.1782707 429 \n13. Yamazaki K, Kashimoto T, Morita M, Kado T, Matsuda K, Yamasaki M, et al. 430 \n2019. Identification of in vivo essential genes of Vibrio vulnificus  for 431 \nestablishment of wound infection by signature-tagged mutagenesis. Front 432 \nMicrobiol  10:123. https://doi.org/10.3389/fmicb.2019.00123 433 \n14. Yamamoto M, Kashimoto T, Tong P, Xiao J, Sugiyama M, Inoue M, et al. 434 \n2015. Signature-tagged mutagenesis of Vibrio vulnificus . J Vet Med Sci  435 \n77:823 –828. https://doi.org/10.1292/jvms.14-0655 436 \n15. Kashimoto T, Yamazaki K, Kado T, Matsuda K, Ueno S. 2021. MukB is a 437 \ngene necessary for rapid proliferation of Vibrio vulnificus  in the systemic 438 \ncirculation but not at the local infection site in the mouse wound infection 439 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n26 \n \nmodel. Microorganisms  9:934. 440 \nhttps://doi.org/10.3390/microorganisms9050934 441 \n16. Kado T, Kashimoto T, Yamazaki K, Matsuda K, Ueno S. 2019. Accurate 442 \nprediction of anti-phagocytic activity of Vibrio vulnificus by measurement of 443 \nbacterial adherence to hydrocarbons. APMIS 127:80 –86. 444 \nhttps://doi.org/10.1111/apm.12910 445 \n17. Yamazaki K, Kashimoto T, Kado T, Yoshioka K, Ueno S. 2022. Increased 446 \nvascular permeability due to spread and invasion of Vibrio vulnificus  in the 447 \nwound infection exacerbates potentially fatal necrotizing disease. Front 448 \nMicrobiol  13:849600. https://doi.org/10.3389/fmicb.2022.849600 449 \n18. Kim SY, Lee SE, Kim YR, Kim CM, Ryu PY, Choy HE, et al. 2003. 450 \nRegulation of Vibrio vulnificus  virulence by the LuxS quorum-sensing system. 451 \nMol Microbiol  48:1647 –1664. 452 \n19. Shao CP, Lo HR, Lin JH, Hor LI. 2011. Regulation of cytotoxicity by 453 \nquorum-sensing signaling in Vibrio vulnificus  is mediated by SmcR, a 454 \nrepressor of hlyU . J Bacteriol  193:2557 –2565. 455 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n27 \n \n20. Gauthier JD, Jones MK, Thiaville P, Joseph JL, Swain RA, Krediet CJ, et al. 456 \n2010. Role of GacA in virulence of Vibrio vulnificus . Microbiology 156:3722 –457 \n3733. 458 \n21. Choi G, Choi SH. 2022. Complex regulatory networks of virulence factors in 459 \nVibrio vulnificus . Trends Microbiol  30:1109 –1121. 460 \nhttps://doi.org/10.1016/j.tim.2022.05.009 461 \n22. Hammer BK, Bassler BL. 2003. Quorum sensing controls biofilm formation in 462 \nVibrio cholerae . Mol Microbiol  50:101 –114. 463 \n23. Park JK, Wang LX, Roseman S. 2002. Isolation of a glucosamine-specific 464 \nkinase, a unique enzyme of Vibrio cholerae . J Biol Chem  277:15573 –15578. 465 \n24. Ran L, Wang X, He X, Guo R, Wu Y, Zhang P, Zhang XH. 2023. Genomic 466 \nanalysis and chitinase characterization of Vibrio harveyi  WXL538: insight 467 \ninto its adaptation to the marine environment. Front Microbiol  14:1121720. 468 \nhttps://doi.org/10.3389/fmicb.2023.1121720  469 \nAcknowledgements  470 \nThe authors thank ChatGPT (OpenAI) and the Nature Research Editing Service for 471 \nassistance with English language editing and manuscript polishing. These tools 472 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n28 \n \nwere used solely to improve clarity and readability of the text and did not 473 \ninfluence the study design, data analysis, or interpretation of results. 474 \nThis work was supported by Grants- in-Aid for Scientific Research (KAKENHI ) 475 \nfrom the Japan Society for the Promotion of Science (JSPS) , Grant Numbers 476 \n19K15979 and 22K14998 awarded to Kohei Yamazaki and 18H02350 awarded to 477 \nTakashige Kashimoto. 478 \nTable 479 \nTable 1. STM-identified genes selected for phenotypic characterization.  480 \nSTM ID \nNo. \nLocus  Gene  Predicted function / category  Reference  \n1/211  VV1_1953  cheY  Chemotaxis signaling  13 \n3 VV1_1937  fliH  Flagellar assembly  13 \n6 VV1_2287  — SM-20–related protein  This study  \n21 VV1_0056  — Predicted membrane protein  This study  \n22 VV1_1300  motX  Polar flagellar motor  13 \n23 VV1_0065  — Transcriptional regulator  This study  \n27 VV1_3091  luxO  \nQuorum -regulated response \nregulator  \nThis study  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n29 \n \n28/233  VV1_0388  — Hypothetical protein  This study  \n29 VV1_3111  — Alcohol dehydrogenase  This study  \n33 VV1_2145  mukB  Chromosome partitioning  13,15 \n34/268  VV1_1573  barA  Hybrid sensory histidine kinase  This study  \n35 VV1_1942  fliM  Flagellar motor switch  This study  \n44 VV1_1935  fliF  Flagellar basal body  This study  \n45 VV2_1016  iutA  Siderophore receptor  13 \n47 VV1_1940  fliK  Flagellar hook -length control  13 \n50 VV1_0312  pomA  Flagellar motor protein  13 \n54 VV2_0147  cspA  Cold -shock / stress response  This study  \n57 VV2_1211  — Predicted membrane protein  This study  \n62 VV2_0122  helD  DNA helicase IV  This study  \n66 VV1_0778  — Glycosyltransferase  13 \n70 VV1_1667  gpsK  Amino -sugar metabolism  13 \n85 VV1_0055  — Putative transmembrane protein  This study  \n107 VV1_1958  cheW  Chemotaxis signaling  13 \n109 VV2_0101  — Lipase -related protein  This study  \n138 VV1_0220  flgH  Flagellar  L-ring protein  This study  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n30 \n \n156 VV1_0501  cstA  Carbon starvation protein  This study  \n160 VV1_1454  — Possible exported protein  This study  \n220 VV1_0375  parA  Chromosome segregation  This study  \n239 VV1_0223  flgE  Flagellar hook protein  This study  \n266 VV1_0219  flgI  Flagellar P -ring protein  This study  \n271 VV1_0692  rpoN  Sigma -54 transcription factor  This study  \n290 VV1_2886  — Hypothetical protein  This study  \n320 VV2_0146  rnb Exoribonuclease II  This study  \n346 VV1_1950  flhF  Flagellar biosynthesis  This study  \n361 VV1_3222  aat N-end rule protein modification  This study  \n370 VV1_1157  ftsX  Cell division protein  This study  \n373 VV1_1930  fliS  Flagellar chaperone  This study  \nNote. Several loci ( cheY , barA , and VV1_0388) were independently identified \nmultiple times in the STM screen, supporting their relevance to in vivo  fitness.  \nFigure legends  481 \nFigure 1. Swimming motility of STM -derived mutants.  482 \nSwimming motility of WT strain and STM -derived mutants assessed on soft -agar 483 \nplates. Images show radial expansion following incubation under identical 484 \nconditions.  485 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n31 \n \nFigure 2. Colony surface properties of STM -derived mutants.  486 \nColony morphology of WT and STM -derived mutants grown on  LB agar plates. 487 \nRepresentative images illustrate differences in colony opacity and surface 488 \nappearance. The left panel shows colony phenotypes, and the right panel indicates 489 \nthe corresponding mutant identification numbers.  490 \nFigure 3. Bioluminescence imaging  of STM -derived mutants during subcutaneous 491 \ninfection.  492 \nBioluminescent signals of WT and selected STM -derived mutants were monitored 493 \nat the indicated time points following subcutaneous infection. Images were 494 \nacquired using identical imaging parameters.  495 \nFigu re 4. Tolerance of STM -derived mutants to phagocytic stress.  496 \nSurvival of WT and STM -derived mutants following exposure to HL-60-derived 497 \nneutrophil s. Data are presented as percent survival relative to control samples 498 \nincubated under identical conditions in the absence of HL -60 cells.  Statistical 499 \nsignificance was evaluated using the Mann –Whitney U test; * P < 0.05 and 500 \n**P < 0.01.  501 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}