Eleonora García Véscovi

Serratia marcescens is an enteric bacterium that can act as an opportunistic pathogen, with increasing incidence in clinical settings. This is largely due to its ability to express a broad repertoire of virulence factors and acquire antibiotic resistance mechanisms. Consequently, S. marcescens has been designated by the World Health Organization (WHO) as a research priority for the development of alternative antimicrobial strategies. In this study, we identify a previously unrecognized role of the type II secretion system (T2SS) in mediating bacterial competition in the clinical strain S. marcescens RM66262. Phylogenomic analysis reveals that the T2SS is conserved among clinical isolates but absent from most environmental strains, suggesting it confers a selective advantage in host-associated or highly competitive environments. Functional assays demonstrate that the T2SS contributes to both inter- and intraspecies antagonism and acts in concert with the contact-dependent type VI secretion system (T6SS), with both systems contributing additively to bacterial killing through distinct mechanisms. We further show that T2SS expression is positively regulated by the PhoP/PhoQ two-component system through direct binding to its promoter region and is induced in response to competitive cues such as attack by Acinetobacter nosocomialis . Altogether, our findings expand the functional scope of the T2SS beyond classical extracellular virulence, establishing it as a PhoP-regulated system involved in bacterial competition and a promising target for novel anti-virulence strategies. IMPORTANCE: This work uncovers a previously uncharacterized role for the type II secretion system (T2SS) in Serratia marcescens , demonstrating its involvement in both inter- and intraspecies bacterial competition. The finding that the T2SS is conserved among clinical isolates but largely absent in environmental strains suggests that this system confers a selective advantage in host-associated or high-competition settings. Furthermore, we show that T2SS expression is regulated by the PhoP/PhoQ system and induced by interbacterial contact, underscoring its role in adaptive antagonistic responses. By expanding the known functional scope of the T2SS, this study provides new insights into the ecological strategies of opportunistic pathogens and highlights a promising target for anti-virulence therapeutic development.

RNAseq analysis of the Drosophila genome has revealed further immune-induced genes. Two genes initially annotated as lncRNAs, CG44404 ( yulü ) and CG45045 ( shenshu ), are strongly induced. We report here that these two genes actually encode highly related secreted peptides found in the Sophophora subgenus of Drosophila species. We have generated single and double null mutants of these loci and found that the double mutant line did not display any enhanced susceptibility to an immune challenge with a panel of bacterial and fungal pathogens, except for Pseudomonas aeruginosa . We did not observe any increased P. aeruginosa burden in yulü-shenshu mutants, suggesting that the two peptides may not be required for resistance to infection. Rather, we find that they provide a level of protection against Outer Membrane Vesicles (OMVs) purified from either P. aeruginosa or Serratia marcescens culture supernatants. We have recently reported that S. marcescens OMVs induce the paralysis of flies through the induction of apoptosis in at least some neurons. Much of the virulence of these OMVs is mediated by the metalloprotease PrtA. While Yulü/Shenshu do not display any protease inhibition activity, the detection of an association between Yulü and the Drosophila complement thioester-containing protein 2 (Tep2) led to the finding that both Tep2 and Tep4 mutants are sensitive to the injection of PrtA while their overexpression significantly protects wild-type flies from the effects of this protease. Both Tep2 and Tep4 are able to inhibit the activity of PrtA in a thioester- and yulü/shenshu -dependent manner. Thus, these Teps may also function as α2-macroglobulins.

Biofilm formation is crucial for bacterial persistence, requiring precise regulatory mechanisms to transition from motility to sessility. Here, we uncover the role of the metalloprotease PrtA in Serratia marcescens biofilm development and its interaction with flagellar components. Loss of PrtA leads to reduced biofilm biomass, thickness, and viable cell counts, as shown through high-resolution confocal microscopy. The biofilm-deficient phenotype is rescued by wild-type PrtA expression but not by a proteolytically inactive PrtA _E177A mutant, underscoring the essential role of PrtA’s enzymatic activity. Exogenous addition of purified PrtA restores biofilm formation, confirming its enzymatic necessity. Proteomic profiling identified flagellar proteins as primary PrtA targets, with an overrepresentation of flagellar components in prtA mutant biofilms. In addition, PrtA selectively degrades depolymerized flagellar filaments, facilitating biofilm progression by removing excess flagellar material. Transcriptional analysis reveals an inverse expression pattern of flagellar master regulator ( flhDC ) and prtA during biofilm establishment, suggesting a coordinated regulatory axis that suppresses flagellar function while promoting biofilm development. Confocal microscopy at the liquid-air interface shows increased flagellar content in prtA mutant biofilms, supporting PrtA’s role in matrix organization and biofilm integrity. Collectively, these findings establish PrtA as a crucial mediator of flagellar turnover and extracellular proteolysis, linking motility suppression to robust biofilm formation. This work not only advances our understanding of biofilm regulation in S. marcescens but also identifies PrtA as a potential target for novel biofilm control strategies. IMPORTANCE: Biofilms are central to the persistence and pathogenicity of Serratia marcescens , particularly in clinical settings where they contribute to chronic infections and antimicrobial resistance. This study identifies the metalloprotease PrtA as a critical regulator of biofilm development, acting through the selective degradation of flagellar components to mediate the transition from motility to sessility. By demonstrating that PrtA’s proteolytic activity is essential for proper biofilm architecture and viability, and that it directly targets excess flagellar material, we provide mechanistic insight into how biofilm maturation is coordinated with motility suppression. The discovery of an inverse regulatory relationship between prtA and the flagellar master regulator flhDC further supports the existence of a finely tuned system controlling biofilm establishment. Together, these findings enhance our understanding of biofilm regulation in Serratia marcescens , an opportunistic human pathogen increasingly associated with antibiotic resistance, and highlight PrtA as a promising target for novel anti-biofilm strategies.

Serratia marcescens , a member of the Enterobacteriaceae family, is an opportunistic human pathogen and a frequent cause of urinary tract infections. Clinical isolates often exhibit resistance to multiple antibiotics, posing challenges for successful treatment. Understanding its pathogenic mechanisms is crucial for elucidating new potential targets to develop effective therapeutic interventions and manage S. marcescens infections. This work identifies u rea- i nduced l actonase of S erratia (UilS), a lactonase encoded in the S. marcescens RM66262 strain isolated from a patient with a urinary tract infection. The study explores the bacterium’s response to urea, a major component of urine, and its impact on uilS expression. We found that UilS degrades acyl-homoserine lactones (AHL) autoinducers traditionally associated with quorum sensing mechanisms. Surprisingly, UilS is able to degrade self and non-self AHL, exhibiting quorum-quenching activity toward Pseudomonas aeruginosa . We found that LuxR regulates uilS expression that is enhanced in the presence of AHL. In addition, urea-dependent induction of UilS expression is controlled by the transcriptional response regulator CpxR. UilS confers fitness advantage to S. marcescens , especially in the presence of urea, emphasizing the adaptive plasticity of strains to modulate gene expression based on environmental signals and population density. We also discovered a novel bacterial killing capacity of S. marcescens that involves UilS, indicating its importance in the interspecies interaction of Serratia . Finally, we found that a uilS mutant strain displays attenuated colonization in a mouse model of catheter-associated urinary tract infection. uilS is present in clinical but absent in environmental isolates, suggesting an evolutionary adaptation to host-specific selective pressures. IMPORTANCE: This work reveals the acyl-homoserine lactonase u rea- i nduced l actonase of S erratia as a novel virulence factor of Serratia marcescens , unraveling a potential target to develop antimicrobial strategies and shedding light on the complex regulatory network governing pathogenicity and adaptation to host environments.

The upsurge of antimicrobial resistance demands innovative strategies to fight bacterial infections. With traditional antibiotics becoming less effective, anti-virulence agents or pathoblockers, arise as an alternative approach that seeks to disarm pathogens without affecting their viability, thereby reducing selective pressure for the emergence of resistance mechanisms. To elucidate the mechanism of action of compound N′-(thiophen-2-ylmethylene)benzohydrazide (A₁₆B₁), a potent synthetic hydrazone inhibitor against the Salmonella PhoP/PhoQ system, essential for virulence. The measurement of the activity of PhoP/PhoQ-dependent and -independent reporter genes was used to evaluate the specificity of A₁₆B₁ to the PhoP regulon. Autokinase activity assays with either the native or truncated versions of PhoQ were used to dissect the A₁₆B₁ mechanism of action. The effect of A₁₆B₁ on Salmonella intramacrophage replication was assessed using the gentamicin protection assay. The checkerboard assay approach was used to analyse potentiation effects of colistin with the hydrazone. The Galleria mellonella infection model was chosen to evaluate A₁₆B₁ as an in vivo therapy against Salmonella. A₁₆B₁ repressed the Salmonella PhoP/PhoQ system activity, specifically targeting PhoQ within the second transmembrane region. A₁₆B₁ demonstrates synergy with the antimicrobial peptide colistin, reduces the intramacrophage proliferation of Salmonella without being cytotoxic and enhances the survival of G. mellonella larvae systemically infected with Salmonella. A₁₆B₁ selectively inhibits the activity of the Salmonella PhoP/PhoQ system through a novel inhibitory mechanism, representing a promising synthetic hydrazone compound with the potential to function as a Salmonella pathoblocker. This offers innovative prospects for combating Salmonella infections while mitigating the risk of antimicrobial resistance emergence.

Colistin remains one of the last-resort therapies for combating infections caused by multidrug-resistant (MDR) Enterobacterales, despite its adverse nephro- and neuro-toxic effects. This study elucidates the mechanism of action of a non-antibiotic 4-anilinoquinazoline-based compound that synergistically enhances the effectiveness of colistin against Salmonella enterica. The quinazoline sensitizes Salmonella by deactivating intrinsic, mutational, and transferable resistance mechanisms that enable Salmonella to counteract the antibiotic impact colistin, together with an induced disruption to the electrochemical balance of the bacterial membrane. The attenuation of colistin resistance via the combined treatment approach also proves efficacious against E. coli, Klebsiella, and Acinetobacter strains. The dual therapy reduces the mortality of Galleria mellonella larvae undergoing a systemic Salmonella infection when compared to individual drug treatments. Overall, our findings unveil the potential of the quinazoline-colistin combined therapy as an innovative strategy against MDR bacteria.

The overprescription and misuse of classical antimicrobial compounds to treat gastrointestinal or systemic salmonellosis have been accelerating the surge of antibiotic-recalcitrant bacterial populations, posing a major public health challenge. Therefore, alternative therapeutic approaches to treat Salmonella infections are urgently required. To identify and characterize actinobacterial secreted compounds with inhibitory properties against the Salmonella enterica PhoP/PhoQ signal transduction system, crucial for virulence regulation. The methodology was based on a combination of the measurement of the activity of PhoP/PhoQ-dependent and -independent reporter genes and bioguided assays to screen for bioactive inhibitory metabolites present in culture supernatants obtained from a collection of actinobacterial isolates. Analogues of azomycin were used to analyse the functional groups required for the detected bioactivity and Salmonella mutants and complemented strains helped to dissect the azomycin mechanism of action. The tetrazolium dye colorimetric assay was used to investigate azomycin potential cytotoxicity on cultured macrophages. Salmonella intramacrophage replication capacity upon azomycin treatment was assessed using the gentamicin protection assay. Sublethal concentrations of azomycin, a nitroheterocyclic compound naturally produced by Streptomyces eurocidicus, repressed the Salmonella PhoP/PhoQ system activity by targeting PhoP and inhibiting its transcriptional activity in a PhoQ- and aspartate phosphorylation-independent manner. Sublethal, non-cytotoxic concentrations of azomycin prevented Salmonella intramacrophage replication. Azomycin selectively inhibits the activity of the Salmonella virulence regulator PhoP, a new activity described for this nitroheterocyclic compound that can be repurposed to develop novel anti-Salmonella therapeutic approaches.

We demonstrate that in S. marcescens , the transcriptional regulator RamA is autoregulated and also controlled by the PhoP/PhoQ signal transduction system. We have shown that PhoP is able to directly and specifically bind to ramA , mgtE1 , mgtE2 , lpxO1 , and lpxO2 promoter regions.

Unsaturated fatty acids promote Salmonella infection by directly repressing the transmembrane sensor PhoQ.