Phenotypic Characterization of Signature-Tagged Mutants Identifies Physiological Determinants of Vibrio vulnificus Fitness

18 Vibrio vulnificus is an opportunistic marine pathogen that causes severe 19 wound-associated and systemic infections. Following entry into the host, the 20 bacterium must rapidly adapt to host-associated stresses that differ substantially 21 from those encountered in aquatic environments. However, the physiological 22 functions supporting bacterial fitness during infection remain incompletely 23 understood. Previously, we applied signature-tagged mutagenesis (STM) to 24 identify genes required for V. vulnificus survival during host infection, revealing 25 numerous loci that did not correspond to classical toxin-encoding genes. In the 26 present study, we extended this genome-wide screen by linking STM-identified 27 mutations to observable fitness-related phenotypes. Functional annotation revealed 28 enrichment of genes associated with chemotaxis, flagellar motility, regulation, 29 metabolism, and poorly characterized functions. Phenotypic analyses showed that 30 many STM-derived mutants exhibited defects in swimming motility and altered 31 colony surface properties. Bioluminescence imaging further revealed distinct 32 patterns of impaired persistence and dissemination within host tissues, while 33 several mutants displayed increased susceptibility to phagocytic stress in an 34 HL-60-derived neutrophil model. Notably, some regulatory mutants affecting 35 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 3 global signaling pathways exhibited impaired tissue dissemination despite 36 retaining resistance to phagocytic stress, indicating the presence of fitness 37 determinants that operate independently of classical surface-associated or 38 cytotoxic traits. Together, these findings demonstrate that V. vulnificus fitness 39 during infection depends on diverse physiological pathways beyond classical 40 virulence factors, highlighting the value of phenotype-centered analyses for 41 understanding bacterial adaptation in host-associated environments. 42 Importance 43 Vibrio vulnificus causes rapidly progressive wound infections and septicemia, yet 44 the bacterial functions that support fitness within host environments remain 45 incompletely defined. While substantial effort has focused on canonical virulence 46 factors and regulation, increasing evidence suggests that successful infection also 47 depends on broader physiological adaptation. In this study, we link 48 signature-tagged mutagenesis with systematic phenotypic analyses to define 49 physiological determinants of V. vulnificus fitness during host-associated infection. 50 Our results demonstrate that genes involved in motility, regulatory signaling, 51 metabolism, and stress tolerance collectively shape bacterial persistence and 52 dissemination in host tissues. Notably, we identify regulatory and metabolic 53 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 4 determinants that influence fitness independently of classical surface-associated or 54 cytotoxic traits, highlighting noncanonical pathways that contribute to pathogenic 55 success. By integrating genome-wide screening with phenotype-centered analyses, 56 this work advances understanding of how physiological adaptation underpins V. 57 vulnificus infection and provides a framework for studying bacterial fitness 58 alongside established concepts of bacterial virulence. 59

60 Vibrio vulnificus ; host-associated environments ; in vivo fitness; signature-tagged 61 mutagenesis (STM); motility; two-component signaling; amino-sugar metabolism; 62 environmental adaptation; bioluminescence imaging 63

64 The public health relevance of Vibrio vulnificus has increased markedly in recent 65 years. Rising sea surface temperatures associated with global climate change have 66 expanded the geographical range of this marine bacterium, leading to increased 67 case numbers and reports of severe wound-associated infections in regions that 68 were previously unaffected (1 , 2). Because V. vulnificus infection is frequently 69 associated with rapidly progressive necrotizing soft tissue infections and high 70 mortality, particularly following wound exposure, it represents a growing public 71 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 5 health concern worldwide (3 –8). As opportunities for environmental exposure 72 continue to grow, there is an increasing need to understand how V. vulnificus 73 adapts to host-associated environments following entry into the human body. 74 Vibrio vulnificus is a marine bacterium that opportunistically invades host tissues 75 through wounds, where it encounters physicochemical constraints, nutrient 76 limitation, and host-derived biotic pressures that differ substantially from those in 77 external aquatic environments. Successful proliferation in soft tissues is known to 78 depend on several bacterial traits that support survival under these stresses. The 79 RTX toxin has been widely reported to exert cytotoxic effects on host immune 80 cells, particularly neutrophils, thereby impairing early innate immune defenses (9, 81 10). In addition, the capsular polysaccharide contributes to resistance against 82 phagocytosis, promoting bacterial persistence in host environments (11). 83 Chemotaxis and flagellar motility further enable bacterial migration and spatial 84 expansion within infected tissues, facilitating access to niches permissive for 85 growth ( 12). 86 Although these traits are established contributors to pathogenicity, they represent 87 only a subset of the bacterial functions required for survival in complex 88 host-associated environments. Increasing evidence from genome-wide analyses 89 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 6 indicates that many genes essential for in vivo fitness do not correspond to 90 classical virulence determinants but instead encode physiological functions 91 involved in regulation, motility, and metabolism ( 13-16). These findings suggest 92 that environmental adaptation, rather than toxin-mediated damage alone, plays a 93 central role during early stages of infection. 94 Previously, we applied signature-tagged mutagenesis (STM) to identify genes 95 required for V. vulnificus fitness during wound infection (13). That study 96 generated a genome-wide map of candidate loci contributing to survival within 97 host tissues and revealed that many STM-identified genes were not canonical 98 virulence factors. Instead, a large proportion of the recovered loci encoded 99 components of motility systems, regulatory elements, metabolic enzymes, or 100 proteins of unknown function, supporting the idea that physiological adaptation is 101 critical for persistence in vivo. 102 In the present study, we extend this work by focusing on phenotypic 103 characterization of representative STM-identified mutants. To directly link STM 104 selection with observable fitness-related traits, we employed in vivo 105 bioluminescence imaging to monitor bacterial persistence and dissemination in 106 real time, together with assays measuring tolerance to phagocyte-associated stress. 107 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 7 This approach allows evaluation of how specific physiological pathways 108 contribute to environmental fitness in host tissues without addressing the 109 molecular mechanisms underlying individual virulence factors. 110 Using this strategy, we show that mutants defective in motility ( motX ), regulatory 111 signaling ( barA and luxO ), and amino-sugar metabolism ( gpsK ) exhibit reduced in 112 vivo fitness and diminished tolerance to phagocytic stress. Together, these 113 findings update and extend our previous STM analysis by integrating functional 114 phenotyping and real-time imaging, providing insight into the physiological 115 systems that support V. vulnificus adaptation to host-associated environments. 116

117 Animals and Ethics Statement 118 Five -week -old female C57BL/6 or BALB/c mice (Charles River Laboratories 119 Japan) were housed under SPF conditions with ad libitum access to food and water, 120 following a 12:12 h light -dark cycle. All animal experiments were approved by the 121 Institutional Animal Care and Use Committee of Kitasato University, in 122 accordance with JALAS guidelines, consistent with our previous reports 123 (Approval No. 19-218). 124 Bacterial Strains 125 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 8 Vibrio vulnificus CMCP6 (clinical isolate) was used as the wild-type (WT) strain. 126 Transposon mutants were generated as described previously (13). Briefly, 127 Escherichia coli BW19795 carrying the signature-tagged mini-Tn5Km2 128 transposon on the suicide vector pUT was used as the donor strain for conjugation. 129 Transposons were introduced into Vibrio vulnificus by biparental mating, and 130 transconjugants were selected on LB agar plates containing kanamycin under 131 appropriate conditions. For genetic complementation, the target gene was 132 amplified by PCR and cloned into the low-copy-number plasmid pACYC184 using 133 In-Fusion cloning reactions (Clontech, TaKaRa, Shiga, Japan), according to th e 134 manufacturer’s instructions. The resulting complementing plasmid was introduced 135 into V. vulnificus by electroporation. Transformants were selected on LB agar 136 plates containing chloramphenicol (10 µg/ml) and incubated overnight at 37°C. 137 All strains were grown aerobically in Luria –Bertani (LB) broth or on LB agar at 138 37 °C, as described previously. When appropriate, antibiotics were added at the 139 following concentrations: rifampicin 50 µg/ml, kanamycin 100 µg/ml, or 140 ampicillin 100 µg/ml. 141 Growth and Inoculati on Conditions 142 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 9 Overnight cultures were diluted 1:20 into fresh LB medium and incubated for 2 h 143 at 37°C with agitation (163 rpm). Bacterial cells were harvested, washed with PBS 144 containing 0.1% gelatin, and resuspended to the desired concentration. Mice wer e 145 inoculated subcutaneously in the caudal thigh with 1 × 10⁶ CFU of wild -type or 146 mutant strains. Infected mice were monitored at defined time points (3 –12 h 147 postinfection) for clinical signs. 148 Identification of transposon insertion sites 149 The transposon insertion sites were determined using arbitrarily primed PCR as 150 described previously (13). Briefly, genomic DNA was isolated from each 151 transposon mutant, and PCR was performed using primers specific to the 152 transposon together with arbitrary primers targeting the Vibrio vulnificus genome. 153 The resulting PCR products were sequenced, and insertion sites were identified by 154 sequence homology searches against the V. vulnificus genome database. 155 Motility assay 156 Swimming motility was assessed on 0.3% ag ar, representing a low -viscosity 157 environment relevant to tissue and fluid interfaces (12,13). 158 Capsule Opacity Assay (CPS phenotype) 159 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 10 Overnight cultures were spotted onto LB agar and incubated 12 h at 37 °C. Colony 160 translucency/opacity was visually inspected (11,13). 161 In vivo bioluminescence imaging 162 To evaluate persistence and dissemination, WT and mutants were transformed with 163 pXen -13 ( luxCDABE ). Bioluminescent WT and mutant strains were visualized 164 using an IVIS -200 imaging system (PerkinElmer). Mice were ane sthetized with 165 isoflurane, and images were acquired with a fixed exposure time of 1 minute at 3, 166 6, 9, and 12 hours after subcutaneous infection (15, 17). 167 Opsonophagocytic survival assay 168 Neutrophil -like cells were generated by differentiating HL -60 cells with 1.25% 169 dimethyl sulfoxide (DMSO) for 5 days , as described previously (16) . Bacterial 170 cells were incubated with differentiated HL -60 cells at a multiplicity of infection 171 (MOI) of 1:20 in the presence of 10% human serum for 45 min at 37°C with gentle 172 rotation. Following incubation, host cells were lysed by treatment with 0.05% 173 saponin, and samples were serially diluted and plated onto LB agar for 174 enumeration of surviving CFU. Bacterial survival was calculated by comparing 175 CFU recovered after incubation wi th HL -60 cells to CFU recovered from control 176 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 11 samples incubated for the same duration under identical conditions in the absence 177 of HL -60 cells. 178 Statistical analysis 179 All statistical analyses were performed using GraphPad Prism (version 8). 180 Quantitative comparisons of bacterial CFU counts were evaluated using the Mann –181 Whitney U test, as the data did not follow a normal distribution. For all 182 experiments, p values of < 0.05 were considered statistically significant. 183

184 Functional catego rization of STM -identified genes 185 To evaluate the functional characteristics of genes identified by the STM screen, 186 we first examined the distribution of annotated functions among the selected loci. 187 All STM -derived mutants exhibited growth kinetics comparable to WT under 188 standard in vitro culture conditions (data not shown), indicating that the observed 189 attenuation was not attributable to intrinsic growth defects. The STM -attenuated 190 gene set was enriched for genes associated with chemotaxis and flage llar motility, 191 including multiple components of the basal body, motor complex, hook, and 192 chemotactic signaling pathways (Table 1) . In addition to motility -associated genes, 193 the STM -identified loci included regulatory elements, metabolic enzymes, stress 194 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 12 response proteins, and factors involved in chromosome maintenance and cell 195 division (Table 1) . A substantial fraction of the identified genes encoded proteins 196 annotated as hypothetical or with limited functional characterization. 197 Motility defects among STM -identified mutants 198 To assess whether STM -identified mutations were associated with defects in 199 motility -related phenotypes, all mutants listed in Table 1 were subjected to a 200 swimming motility assay as an initial phenotypic screen. WT exhibited robust 201 radial e xpansion on soft -agar plates, whereas multiple STM -derived mutants 202 displayed marked reductions in motility (Fig. 1). 203 As expected, disruptions in genes encoding flagellar structural components and 204 motor -associated proteins , including motX, fliH, fliM, fliF , fliK, flgE, flgH, flgI, 205 and flhF , resulted in severe motility defects (Fig. 1). Mutations in chemotactic 206 signaling proteins ( cheY and cheW ) similarly impaired swimming behavior, 207 consistent with their established roles in directed bacterial movement. In a ddition 208 to these canonical motility -related genes, several mutants carrying insertions in 209 genes not directly annotated as motility components also exhibited reproducible 210 reductions in swimming motility. These included genes encoding a siderophore 211 receptor (iutA ), predicted membrane proteins, lipase -related proteins, exported 212 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 13 proteins, chromosome segregation factors ( parA ), the sigma -54 transcription 213 factor ( rpoN ), and an N -end rule protein modification enzyme ( aat) (Fig. 1). These 214 findings indicate that div erse physiological and regulatory functions contribute 215 indirectly to motility -associated behaviors in V. vulnificus . 216 Capsule -associated colony opacity variations 217 Colony morphology was assessed as an indicator of surface -associated properties 218 potentially relevant to host adaptation. WT formed opaque colonies on LB agar, 219 whereas several STM -derived mutants produced colonies with increased 220 translucency (Fig. 2). Notab ly, mutants carrying insertions in the regulatory 221 signaling gene barA and a gene encoding a glycosyltransferase exhibited a 222 translucent colony phenotype comparable to that observed in the environmental 223 isolate control strain E4 (Fig. 2) . This altered colon y morphology was 224 reproducibly observed in mutants affecting regulatory signaling pathways and 225 glycosylation -related functions. In contrast, STM -derived mutants disrupted in 226 flagellar motility or chemotactic signaling genes retained colony opacity 227 comparabl e to that of the wild -type strain, indicating that the observed 228 translucency phenotype was not a general consequence of STM mutagenesis but 229 was associated with specific functional categories. 230 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 14 Reduced persistence and dissemination in soft tissues among repr esentative 231 STM-derived mutants 232 To determine whether STM -identified mutations impaired bacterial fitness in soft 233 tissues, bioluminescent derivatives of selected mutants were monitored during 234 subcutaneous infection using in vivo bioluminescence imaging (IVIS). WT 235 exhibited progressive dissemination of luminescent signals from the inoculation 236 site, followed by signal attenuation by 12 h postinfection, consistent with active 237 persistence, expansion, and subsequent invasion into deep er soft tissues (Fig. 3) 238 (12, 17). 239 Based on IVIS signal dynamics, STM -derived mutants could be broadly classified 240 into three phenotypic groups. The first group displayed behavior in soft tissues 241 comparable to that of WT. Mutants carrying insertions in VV1_ 0388 and 242 VV1_0778 showed sustained luminescent signals and spatial dissemination similar 243 to those observed for WT, indicating that disruption of these loci did not markedly 244 impair persistence or spread in soft tissues. 245 The second group exhibited severe def ects in dissemination from the infection site. 246 As expected, the motility -deficient mutant motX::Tn failed to spread within soft 247 tissues, consistent with previous observations that bacterial motility is essential 248 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 15 for expansion and dissemination in this infe ction model (12). Similarly, mutants 249 disrupted in regulatory signaling pathways ( luxO::Tn and barA::Tn ) showed 250 restricted luminescent signals that remained localized near the inoculation site, 251 resembling the phenotype of the motility -deficient mutant ( 13). 252 The third group showed limited dissemination accompanied by prolonged 253 persistence of luminescent signals at the infection site. The chemotaxis -deficient 254 mutant cheW::Tn exhibited spread within soft tissues; however, luminescent 255 signals remained detectable over the observation period. This phenotype is 256 consistent with previously described behavior of chemotaxis mutants that retain 257 motility but exhibit impaired invasion into deeper tissue compartments (12). A 258 similar IVIS phenotype was observed for several a dditional STM -derived mutants, 259 including mukB ::Tn , ftsX ::Tn , gpsK ::Tn , VV1_0055::Tn, VV1_0056::Tn, 260 VV2_0147::Tn, VV2_0122 ::Tn , VV1_0501::Tn, and VV2_0146::Tn. 261 Collectively, IVIS analysis revealed distinct phenotypic classes among 262 STM-derived mutants in sof t tissues, reflecting differential defects in bacterial 263 persistence, dissemination within soft tissues, and invasion into deeper tissue 264 compartments. 265 Increased susceptibility of STM -derived mutants to phagocytic stress 266 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 16 Because early host responses to subcutaneous infection involve rapid recruitment 267 of phagocytic cells, STM -derived mutants were examined for tolerance to 268 phagocytic stress using differentiated HL -60 cells as a neutrophil -like cell model. 269 WT demonstrated sub stantial survival following exposure to phagocytic cells (Fig. 270 4). The barA ::Tn mutant exhibited survival comparable to that of WT (Fig. 4), 271 indicating that disruption of barA did not markedly impair resistance to phagocytic 272 stress under these conditions. 273 In contrast, several STM -derived mutants showed significantly reduced survival 274 after incubation with HL -60 cells. Mutants carrying insertions in motX , luxO , 275 VV1_0388, and gpsK displayed pronounced decreases in survival relative to WT 276 (Fig. 4), indicating i ncreased susceptibility to phagocyte -associated stress. These 277 reductions were consistently observed across independent experiments. 278 To confirm that the observed phenotypes were attributable to the respective 279 transposon insertions, complementation analyses were performed. Introduction of 280 the corresponding wild -type genes restored survival to levels comparable to WT, 281 confirming that the increased susceptibility to phagocytic stress resulted from 282 disruption of the targeted loci. 283 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 17

284 In this study, we extended our previous STM -based analysis by integrating 285 targeted phenotypic characterization to define physiological traits associated with 286 Vibrio vulnificus fitness in host -associated environments. By combining STM 287 selection with motilit y assays, evaluation of surface -associated properties, 288 bioluminescence -based imaging in soft tissues, and phagocytic stress assays, we 289 directly linked STM -identified loci to observable fitness -related phenotypes 290 without addressing the molecular mechanisms underlying individual virulence 291 factors. 292 The functional composition of STM -identified genes provides initial validation of 293 the screening strategy. The enrichment of chemotaxis - and flagellar motility –294 associated genes among STM -attenuated mutants is consist ent with the established 295 importance of directed movement for bacterial expansion within soft tissues (Table 296 1) (12, 13). Motility enables bacteria to explore heterogeneous host environments 297 and access niches permissive for growth, and its repeated recovery in STM -based 298 screens supports the view that motility -related functions represent core 299 physiological requirements for fitness during early infection (12). However, the 300 motility -deficient motX mutant retained substantial resistance to phagocytic stress 301 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 18 (Fig . 4). In the HL -60 survival assay, motX ::Tn exhibited a median survival of 302 83.5%, indicating that the absence of motility alone does not render V. vulnificus 303 completely susceptible to phagocytic killing. This residual resistance is likely 304 supported by addi tional virulence -associated traits retained by the motX mutant, 305 such as capsular polysaccharide and RTX toxin production (9,10,12 ). 306 In contrast, genes encoding the RTX toxin were not recovered among 307 STM-selected loci (Table 1) . This absence likely reflects a methodological 308 characteristic of STM -based negative selection screens rather than a lack of 309 contribution of RTX to virulence. Because STM involves pooled infection with 310 multiple mutant strains, secreted virulence factors such as toxins can be 311 functional ly complemented by neighboring bacteria. Under these conditions, 312 mutants defective in toxin production may not exhibit a competitive disadvantage 313 and therefore escape negative selection. Consequently, STM preferentially 314 identifies cell -autonomous physiolog ical functions required for survival and 315 persistence, while diffusible virulence factors are underrepresented (13,14 ). 316 Beyond motility -associated genes, the STM -identified set included regulatory 317 elements, metabolic enzymes, stress response proteins, and f actors involved in 318 chromosome maintenance and cell division (15,16,18-23). Notably, a substantial 319 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 19 fraction of the recovered loci encoded proteins of unknown or poorly 320 characterized function. The recurrence of such genes among STM -attenuated 321 mutants suggests that V. vulnificus relies on additional, incompletely understood 322 physiological processes to adapt to host -associated environments. These findings 323 underscore the value of genome -wide screening approaches for uncovering fitness 324 determinants that are not readily predicted from existing virulence models. 325 Phenotypic analyses revealed that disruptions in motility and chemotaxis strongly 326 impair bacterial migration (Fig. 1), confirming tha t these traits are tightly linked to 327 fitness in soft tissues (Fig. 3). Although luxO and barA mutants retained normal 328 swimming motility in vitro (Fig. 1), both mutants exhibited severe defects in 329 persistence and dissemination within soft tissues (Fig. 3), resembling the 330 phenotype of the motility -deficient motX mutant. This discrepancy indicates that 331 regulatory pathways controlled by LuxO and BarA contribute to fitness in soft 332 tissues through mechanisms that are independent of flagellar motility. Rather than 333 affecting bacterial movement per se, these regulators likely coordinate additional 334 physiological processes required for growth, persistence, o r spatial expansion 335 within host tissues. 336 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 20 Alterations in colony opacity among selected STM mutants further suggest that 337 regulatory and metabolic pathways influence bacterial surface architecture. The 338 glycosyltransferase mutant (VV1_0778::Tn) formed transluc ent colonies, which 339 suggests reduced capsular polysaccharide production (Fig. 2), yet it retained the 340 ability to disseminate within soft tissues as efficiently as WT (Fig. 3). In contrast, 341 the barA ::Tn mutant also exhibited a translucent colony phenotype ( Fig. 2), but 342 IVIS analysis revealed marked defects in tissue dissemination (Fig. 3), despite this 343 mutant retaining resistance to phagocytic stress (Fig. 4). These observations 344 indicate that neither colony opacity nor resistance to phagocytic stress alone c an 345 reliably predict bacterial fitness in soft tissues, suggesting that additional factors 346 independent of capsule production and motility contribute to successful growth 347 and spread in this environment. 348 The IVIS analysis further revealed distinct phenotypic classes among 349 STM-derived mutants, reflecting differential defects in persistence, dissemination, 350 and invasion into deeper tissue compartments (Fig. 3) (12) . In particular, 351 regulatory mutants ( luxO ::Tn and barA ::Tn) exhibited dissemination defects 352 comparab le to those of motility -deficient strains, despite differing phenotypes in 353 other assays. LuxO is a central regulator of quorum -sensing pathways in V. 354 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 21 vulnificus and has been implicated in the control of virulence -associated gene 355 expression, including facto rs linked to RTX toxin regulation ( 18-21). Although 356 RTX genes were not directly identified in the STM screen, impaired regulation of 357 toxin expression or secretion may contribute indirectly to the reduced fitness of 358 luxO mutants in soft tissues. 359 Consistent with the IVIS analysis, several STM -derived mutants displayed 360 increased susceptibility to phagocytic stress in an HL -60-derived neutrophil model 361 (Fig. 4). Reduced survival among motility -, regulatory -, and metabolism -defective 362 mutants indicates that these pathways contribute to tolerance of host -derived 363 biotic pressures encountered during early infection. Notably, barA ::Tn retained 364 resistance to phagocytic stress despite exhibiting impaired dissemination in soft 365 tissues, further supporting the notion that f itness in this environment reflects the 366 integration of multiple physiological traits rather than reliance on a single immune 367 evasion mechanism. 368 GpsK is a glucosamine -specific kinase that catalyzes the conversion of 369 glucosamine to glucosamine -6-phosphate, r epresenting the initial step of amino 370 sugar utilization in Vibrio species ( 23). This pathway constitutes a central 371 component of chitin -derived nutrient metabolism and has been implicated in 372 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 22 environmental adaptation of marine Vibrios (24). Although disrupti on of gpsK did 373 not result in detectable growth impairment under in vitro conditions, loss of this 374 enzyme may restrict metabolic flexibility in host -associated environments, where 375 accessible carbon and nitrogen sources are limited. Such constraints could 376 compromise bacterial fitness in soft tissues, contributing to the delayed tissue 377 invasion and increased susceptibility to phagocytic stress observed for the 378 gpsK ::Tn mutant. These findings suggest that gpsK supports in vivo fitness not by 379 enhancing basal gro wth capacity but by enabling metabolic adaptation to 380 nutrient -limited and stress -rich host environments. 381 Taken together, the results of this study indicate that Vibrio vulnificus fitness in 382 host -associated environments is governed by a network of physiolog ical functions 383 encompassing motility, regulatory signaling, metabolism, and stress tolerance. 384 Many of the identified determinants do not correspond to classical virulence 385 factors but instead contribute to bacterial adaptation under host -imposed 386 constraints . By linking STM -based selection with phenotypic outcomes, this work 387 provides further insight into how environmental adaptation underpins bacterial 388 persistence during early stages of infection and highlights the value of 389 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 23 phenotype -centered approaches for d issecting in vivo fitness beyond canonical 390 virulence paradigms. 391

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470 The authors thank ChatGPT (OpenAI) and the Nature Research Editing Service for 471 assistance with English language editing and manuscript polishing. These tools 472 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 28 were used solely to improve clarity and readability of the text and did not 473 influence the study design, data analysis, or interpretation of results. 474 This work was supported by Grants- in-Aid for Scientific Research (KAKENHI ) 475 from the Japan Society for the Promotion of Science (JSPS) , Grant Numbers 476 19K15979 and 22K14998 awarded to Kohei Yamazaki and 18H02350 awarded to 477 Takashige Kashimoto. 478 Table 479 Table 1. STM-identified genes selected for phenotypic characterization. 480 STM ID No. Locus Gene Predicted function / category Reference 1/211 VV1_1953 cheY Chemotaxis signaling 13 3 VV1_1937 fliH Flagellar assembly 13 6 VV1_2287 — SM-20–related protein This study 21 VV1_0056 — Predicted membrane protein This study 22 VV1_1300 motX Polar flagellar motor 13 23 VV1_0065 — Transcriptional regulator This study 27 VV1_3091 luxO Quorum -regulated response regulator This study .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 29 28/233 VV1_0388 — Hypothetical protein This study 29 VV1_3111 — Alcohol dehydrogenase This study 33 VV1_2145 mukB Chromosome partitioning 13,15 34/268 VV1_1573 barA Hybrid sensory histidine kinase This study 35 VV1_1942 fliM Flagellar motor switch This study 44 VV1_1935 fliF Flagellar basal body This study 45 VV2_1016 iutA Siderophore receptor 13 47 VV1_1940 fliK Flagellar hook -length control 13 50 VV1_0312 pomA Flagellar motor protein 13 54 VV2_0147 cspA Cold -shock / stress response This study 57 VV2_1211 — Predicted membrane protein This study 62 VV2_0122 helD DNA helicase IV This study 66 VV1_0778 — Glycosyltransferase 13 70 VV1_1667 gpsK Amino -sugar metabolism 13 85 VV1_0055 — Putative transmembrane protein This study 107 VV1_1958 cheW Chemotaxis signaling 13 109 VV2_0101 — Lipase -related protein This study 138 VV1_0220 flgH Flagellar L-ring protein This study .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 30 156 VV1_0501 cstA Carbon starvation protein This study 160 VV1_1454 — Possible exported protein This study 220 VV1_0375 parA Chromosome segregation This study 239 VV1_0223 flgE Flagellar hook protein This study 266 VV1_0219 flgI Flagellar P -ring protein This study 271 VV1_0692 rpoN Sigma -54 transcription factor This study 290 VV1_2886 — Hypothetical protein This study 320 VV2_0146 rnb Exoribonuclease II This study 346 VV1_1950 flhF Flagellar biosynthesis This study 361 VV1_3222 aat N-end rule protein modification This study 370 VV1_1157 ftsX Cell division protein This study 373 VV1_1930 fliS Flagellar chaperone This study Note. Several loci ( cheY , barA , and VV1_0388) were independently identified multiple times in the STM screen, supporting their relevance to in vivo fitness. Figure legends 481 Figure 1. Swimming motility of STM -derived mutants. 482 Swimming motility of WT strain and STM -derived mutants assessed on soft -agar 483 plates. Images show radial expansion following incubation under identical 484 conditions. 485 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 18, 2025. ; https://doi.org/10.64898/2025.12.18.695119doi: bioRxiv preprint 31 Figure 2. Colony surface properties of STM -derived mutants. 486 Colony morphology of WT and STM -derived mutants grown on LB agar plates. 487 Representative images illustrate differences in colony opacity and surface 488 appearance. The left panel shows colony phenotypes, and the right panel indicates 489 the corresponding mutant identification numbers. 490 Figure 3. Bioluminescence imaging of STM -derived mutants during subcutaneous 491 infection. 492 Bioluminescent signals of WT and selected STM -derived mutants were monitored 493 at the indicated time points following subcutaneous infection. Images were 494 acquired using identical imaging parameters. 495 Figu re 4. Tolerance of STM -derived mutants to phagocytic stress. 496 Survival of WT and STM -derived mutants following exposure to HL-60-derived 497 neutrophil s. Data are presented as percent survival relative to control samples 498 incubated under identical conditions in the absence of HL -60 cells. Statistical 499 significance was evaluated using the Mann –Whitney U test; * P < 0.05 and 500 **P < 0.01. 501 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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