microLife

Patients with Crohns disease exhibit abnormal colonization of the intestine by proteobacteria, and among these bacteria, the adherent invasive E. coli (AIEC) family. They are predominant in the mucus, adhere to epithelial cells, colonize them and survive inside macrophages. We recently demonstrated that the acclimation of the AIEC strain LF82 to phagolysosomal stress requires stringent and SOS responses. Such adaptation involves a long lag phase in which many LF82 cells become antibiotic tolerant. Later during infection, they proliferate in vacuoles and form colonies harboring dozens of LF82 bacteria. In the present work, we investigated the mechanism sustaining this phase of growth. We found that intracellular LF82 produced an extrabacterial matrix composed of exopolysaccharides and amyloid fibers that surrounded each individual LF82 cell. This matrix acts as a biofilm and controls the formation of LF82 intracellular bacterial communities (IBCs) inside phagolysosomes for several days post infection. Using genomics assays, we characterized the gene set involved in IBCs formation and revealed the crucial role played by a pathogenicity island presents in the genome of most AIEC strains in this process. Iron capture, by the yersiniabactin system encoded by this pathogenicity island, is essential to form IBC and LF82 survival within macrophages. These results demonstrate that AIEC have developed a sophisticated strategy to establish their replicative niche within macrophages, which might have implications for envisioning future antibacterial strategies for Crohns disease.

Bacteria can rapidly tune their physiology and metabolism to adapt to environmental fluctuations. In particular, they can adapt their lifestyle to the close proximity of other bacteria or presence of different surfaces. However, whether these interactions trigger transcriptomic responses is poorly understood. We used a specific set up of E. coli strains expressing native or synthetic adhesins mediating bacterial aggregation to study the transcriptomic changes of aggregated compared to non-aggregated bacteria. Our results show that following aggregation, bacteria exhibit a core response independent of the adhesin type, with differential expression of 56.9% of the coding genome, including genes involved in stress response and anaerobic lifestyle. Moreover, when aggregates were formed via a naturally expressed E. coli adhesin (Antigen 43), the transcriptomic response of the bacteria was more exaggerated compared to aggregates formed via a synthetic adhesin. This suggests that the response to aggregation induced by native E. coli adhesins could have been finely tuned during bacterial evolution. Our study therefore provides insights on the effect of self-interaction in bacteria and allows a better understanding of why bacterial aggregates exhibit increased stress tolerance. ImportanceFormation of bacterial aggregates has an important role in both clinical and ecological contexts. Although these structures have been previously shown to be more resistant to stressful conditions, the genetic basis of this stress tolerance associated with the aggregate lifestyle is poorly understood. Surface sensing mediated by different adhesins can result in varying changes on bacterial physiology. However, whether adhesin-adhesin interactions as well as the type of adhesin mediating aggregation affects bacterial cell physiology is unknown. By sequencing the transcriptomes of aggregated and non-aggregated cells expressing native or synthetic adhesins, we characterized the effects of aggregation and adhesin type on E. coli physiology.

CRISPR-Cas systems in prokaryotes utilize spacers, segments of DNA acquired from invading phages, to guide immune defense mechanisms. This study investigates the evolution of CRISPR repertoire size by examining its relationships with repeat length, terminal repeat polymorphism, and structural stability in 1,958 bacterial genomes, identifying 5,465 CRISPR arrays. Using CRISPRCasFinder for annotation and RNAfold for predicting RNA secondary structures, we found significant variation in array characteristics. Long-repeat arrays ([&ge;]38 bp) showed a significant positive correlation between terminal repeat polymorphism and CRISPR spacer number, a correlation absent in short-repeat arrays (<38 bp), suggesting longer repeats facilitate recombination and spacer loss. Additionally, a negative correlation between repeat length and spacer number across all arrays indicates that longer repeats may accelerate spacer loss. Furthermore, our results show that immune demand significantly influences the evolution of spacer number. Larger CRISPR repertoires correlate with conserved repeat sequences and stable secondary structures, vital for functional arrays under continuous selective pressure. Comparing functional and obsolete CRISPR arrays (orphan arrays in genomes lacking Cas genes) revealed that obsolete arrays have fewer spacers and lower repeat consistency, indicating a degenerative state. By elucidating the factors that shape CRISPR memory size evolution, this research offers strategies to enhance bacterial defenses, mitigate resistance, and improve applications in gene editing and therapeutics.

Cellulose is a major component of the Salmonella biofilm extracellular matrix and it is considered an antivirulence factor because it interferes with Salmonella survival inside macrophages and virulence in mice. Its synthesis is stimulated by CsgD, the master regulator of biofilm extracellular matrix formation in enterobacteria, which in turn is under the control of MlrA, a MerR-like transcription factor. In this work we identified a SPI-2 encoded Salmonella-specific transcription factor homolog to MlrA, MlrB, that represses transcription of its downstream gene, STM1389, also known as orf319, and of csgD inside host cells. MlrB is induced in laboratory media mimicking intracellular conditions and inside macrophages, and it is required for intramacrophage survival. An increased expression of csgD is observed in the absence of MlrB inside host cells. Interestingly, inactivation of the CsgD-controlled cellulose synthase coding-gene, bcsA, restored intramacrophage survival to rates comparable to wild type bacteria in the absence of MlrB. These data indicate that MlrB represses CsgD expression inside host cells and in consequence activation of the cellulose synthase. Our findings provide a novel link between biofilm formation and Salmonella virulence.

Lipopolysaccharide is essential for most Gram-negative bacteria as it is a main component of the outer membrane. In the pathogen Brucella abortus, smooth lipopolysaccharide containing the O-antigen is required for virulence. Being part of the Rhizobiales, Brucella spp. display unipolar growth and lipopolysaccharide was shown to be incorporated at the active growth sites, i.e. the new pole and the division site. By localizing proteins involved in the lipopolysaccharide transport across the cell envelope, from the inner to the outer membrane, we show that the lipopolysaccharide incorporation sites are determined by the inner membrane complex of the lipopolysaccharide transport system. Moreover, we identify the main O-antigen ligase of Brucella spp involved in smooth lipopolysaccharide synthesis. Altogether, our data highlight a new layer of spatiotemporal organization of the lipopolysaccharide biosynthesis pathway and identify a new class of bifunctional O-antigen ligases.

CRISPR-Cas systems are best known for their role in adaptive immunity, but emerging evidence suggests broader regulatory functions. Here, we show that the CRISPR-Cas system acts as a serovar-specific regulator of stress adaptation in Salmonella enterica, exerting opposing effects in host-restricted (S. Typhi) and broad-host-range (S. Typhimurium) serovars. In S. Typhi, CRISPR-Cas system deletion reduces acid and bile tolerance by impairing envelope integrity and repressing key stress-response regulators (envZ, cadB, phoPQ, lexA, ruvB, wecD), while increasing resistance to cationic antimicrobial peptides via pmr activation and reduced oxidative damage. Conversely, CRISPR-Cas system loss in S. Typhimurium enhances acid survival-partly through speF upregulation but increases sensitivity to antimicrobial peptides. Spacer-1 of S. Typhi CRISPR-I array as the main regulator of gene expression, and its reintroduction partially restored stress tolerance, supporting spacer-dependent control of physiological pathways. These findings establish the CRISPR-Cas system as a non-canonical, spacer-dependent regulator of stress response networks in S. enterica, revealing its contribution to the evolutionary divergence of survival strategies between S. Typhi and S. Typhimurium.

Patients with Crohns disease exhibit abnormal colonization of the intestine by Proteobacteria, particularly the adherent-invasive Escherichia coli (AIEC) group. These bacteria are predominant in the mucus, adhere to epithelial cells, colonize them, and survive inside macrophages. We recently demonstrated that the acclimation of strain LF82 to phagolysosomal stress occurs in two distinct steps: first, a replication halt producing stress- tolerant persisters, and second, a replication phase that leads to the formation of Intracellular Bacterial Communities (IBC) organized with a biofilm-like matrix. Given the significant genomic diversity among strains with the AIEC phenotype, we conducted a comparative analysis of the genomes and macrophage colonization characteristics of 13 AIEC strains collected from patients during a clinical study conducts by the CHU of Clermont-Ferrand. Our results demonstrate that IBCs serve as replicative niches for all AIEC strains within macrophages. However, these strains form IBCs using different strategies, including varying levels of phagosome detoxification, distinct biofilm characteristics, and diverse macrophage responses. Our study reveals a strong positive correlation between vacuole acidification and persister induction that explains intracellular survival of the different strains. In addition, we revealed distinct AIEC dissemination strategies outside macrophages, which may contribute to the propagation of inflammation in the human host. These findings highlight that research on pathogens and pathobionts with plastic genomes should not rely solely on a few laboratory models.

The bacterial cell envelope is an essential structure that protects the cell from environmental threats, while simultaneously serving as communication interface and diffusion barrier. Therefore, maintaining cell envelope integrity is of vital importance for all microorganisms. Not surprisingly, evolution has shaped conserved protection networks that connect stress perception, transmembrane signal transduction and mediation of cellular responses upon cell envelope stress. The phage shock protein (PSP) stress response is one of such conserved protection networks. Most of the knowledge about the Psp response comes from studies in the Gram-negative model bacterium, Escherichia coli where the Psp system consists of several well-defined protein components. Homologous systems were identified in representatives of Proteobacteria, Actinobacteria, and Firmicutes; however, the Psp system distribution in the microbial world remains largely unknown. By carrying out a large-scale, unbiased comparative genomics analysis, we found components of the Psp system in many bacterial and archaeal phyla and demonstrated that the PSP system deviates dramatically from the proteobacterial prototype. Two of its core proteins, PspA and PspC, have been integrated in various (often phylum-specifically) conserved protein networks during evolution. Based on protein sequence and gene neighborhood analyses of pspA and pspC homologs, we built a natural classification system of PSP networks in bacteria and archaea. We performed a comprehensive in vivo protein interaction screen for the PSP network newly identified in the Gram-positive model organism Bacillus subtilis and found a strong interconnected PSP response system, illustrating the validity of our approach. Our study highlights the diversity of PSP organization and function across many bacterial and archaeal phyla and will serve as foundation for future studies of this envelope stress response beyond model organisms.

Natural competence for genetic transformation, considered as one of the three main mechanisms leading to horizontal gene transfer in bacteria, is able to promote evolution, through genomic plasticity, and foster antibiotic resistance and virulence factors spreading. Conserved machineries and actors required to perform genetic transformation have been shown to accumulate at different cellular localizations depending on the model organism considered. Here, we show in the human pathogen Staphylococcus aureus that DNA binding, uptake and recombination are spatially and temporally coordinated to ensure S. aureus genetic transformation. We also reveal that localization of genetic transformation proteins is dynamic and preferentially occurs in the vicinity of the division septum. We finally propose that S. aureus competent cells would initiate and then block cell division to ensure the success of genetic transformation before the final constriction of the cytokinetic ring.

NLRs (Nod-like receptors) are intracellular receptors regulating immunity, symbiosis, non-self recognition and programmed cell death in animals, plants and fungi. Several fungal NLRs employ amyloid signaling motifs to activate downstream cell-death inducing proteins. Herein, we identify in Archaea and Bacteria, short sequence motifs that occur in the same genomic context as fungal amyloid signaling motifs. We identify 10 families of bacterial amyloid signaling sequences (we term BASS), one of which (BASS3) is related to mammalian RHIM and fungal PP amyloid motifs. We find that BASS motifs occur specifically in bacteria forming multicellular structures (mainly in Actinobacteria and Cyanobacteria). We analyze experimentally a subset of these motifs and find that they behave as prion forming domains when expressed in a fungal model. All tested bacterial motifs also formed fibrils in vitro. We analyze by solid-state NMR and X-ray diffraction, the amyloid state of a protein from Streptomyces coelicolor bearing the most common BASS1 motif and find that it forms highly ordered non-polymorphic amyloid fibrils. This work expands the paradigm of amyloid signaling to prokaryotes and underlies its relation to multicellularity.

CRISPR-Cas systems endow bacteria and archaea with adaptive immunity against mobile genetic elements, playing a fundamental role in shaping microbial communities. Many organisms harbor more than one CRISPR-Cas system, and little is known about whether and how they are differentially regulated, in many instances due to the impossibility of studying CRISPR immunity in native hosts. Here we studied the regulation of endogenous type II-B and type V-A CRISPR-Cas systems in opportunistic human pathogen Francisella novicida U112. Fluorescence microscopy and transcriptomics experiments revealed that while the type II-B system is constitutively expressed, the type V-A CRISPR-Cas system is differentially expressed at stationary phase and high cell density. Using mass spectrometry and genetics we identified MtvS as a factor required for the differential expression of the type V-A CRISPR-Cas locus. Surprisingly, MtvS-dependent expression of the type V-A CRISPR-Cas system at high cell density is linked to a quorum sensing-like behavior. In addition, MtvS modulates transcription of many genes in stationary phase, some of which are required for Francisella virulence. Pull-down experiments revealed MtvS interacts with the {beta} subunit of the RNA polymerase and therefore may constitute a noncanonical alternative sigma factor involved in the regulation of the expression of CRISPR loci and other genes.

In prokaryotes, CRISPR-Cas systems store memories of past infections in the form of spacers integrated into CRISPR arrays. When associated with type III CRISPR-Cas systems, Reverse transcriptase-Cas1 fusion proteins (RT-Cas1) enable these defense systems to acquire spacers from RNA sources. However, despite the specific targeting of RNA by the Cas13-containing type VI CRISPR-Cas systems, there is no evidence of RNA-origin spacer acquisition. Using computational analyses, we recently reported the association of RT-Cas1 fusion proteins with type VI-A systems. In this study, we found that RT-Cas1 fusion proteins were also associated with complete type VI-B systems in bacteria from gut metagenomes, constituting a variant system that harbors a linked CorA-encoding locus in addition to the CRISPR array and adaptation RT-Cas1/Cas2 module. By combining in vitro and in vivo experiments, we demonstrated that type VI RT-CRISPR systems are functional for spacer acquisition and CRISPR array processing, and that the associated RT enables spacer acquisition from RNA molecules, thus demonstrating that the system is capable of functioning independently of other in-trans systems. These findings highlight the importance of RTs in RNA-targeting CRISPR-Cas systems, suggesting a potential defense mechanism against RNA-based invaders in specific environments.

Type VI secretion systems (T6SS) are common bacterial contractile injection systems that inject toxic "effector" proteins into neighboring cells. Effector discovery is generally done manually, and computational approaches used for effector discovery depend on genetic linkage to T6SS genes and/or sequence similarity to known effectors. We bioinformatically investigated T6SS in more than 11,832 genomes of Gram negative bacteria. We found that T6SS encoding bacteria are host-associated and pathogenic, enriched in specific human and plant tissues, while depleted in marine, soil, and engineered environments. Analysis of T6SS cores with C-terminal domains ("evolved" cores) showed "evolved" HCP are rare, overwhelmingly encoded in orphan operons, and are largely restricted to Escherichia. Using the wealth of data generated from our bioinformatic analysis, we developed two algorithms for large-scale discovery of T6SS effector proteins (T6Es). We experimentally validated ten putative antibacterial T6SS effector proteins and one cognate immunity gene from a diverse species. This study provides a systematic genomic perspective of the role of the T6SS in nature, a thorough analysis of T6E evolution and genomic properties, and discovery of a large number of candidate T6Es using new approaches.

The type VI secretion system (T6SS) is a contact-dependent contractile multiprotein apparatus widely distributed in Gram-negative bacteria. These systems can deliver different effector proteins into target bacterial and/or eukaryotic cells, contributing to the environmental fitness and virulence of many bacterial pathogens. Salmonella harbors five different T6SSs encoded in different genomic islands. The T6SS encoded in Salmonella Pathogenicity Island 6 (SPI-6) contributes to Salmonella competition with the host microbiota and its interaction with infected host cells. Despite its relevance, information regarding the total number of effector proteins encoded within SPI-6 and its distribution among different Salmonella enterica serotypes is limited. In this work, we performed bioinformatic and comparative genomics analyses of the SPI-6 T6SS gene cluster to expand our knowledge regarding the T6SS effector repertoire and the global distribution of these effectors in Salmonella. The analysis of a curated dataset of 60 Salmonella enterica genomes from the Secret6 database revealed the presence of 23 novel putative T6SS effector/immunity protein (E/I) modules. These effectors were concentrated in the variable regions 1 to 3 (VR1-3) of the SPI-6 T6SS gene cluster. VR1-2 were enriched in candidate effectors with predicted peptidoglycan hydrolase activity, while VR3 was enriched in candidate effectors of the Rhs family with C-terminal extensions with predicted DNase, RNase, deaminase, or ADP-ribosyltransferase activity. A global analysis of known and candidate effector proteins in Salmonella enterica genomes from the NCBI database revealed that T6SS effector proteins are differentially distributed among Salmonella serotypes. While some effectors are present in over 200 serotypes, others are found in less than a dozen. A hierarchical clustering analysis identified Salmonella serotypes with distinct profiles of T6SS effectors and candidate effectors, highlighting the diversity of T6SS effector repertoires in Salmonella enterica. The existence of different repertoires of effector proteins suggests that different effector protein combinations may have a differential impact on the environmental fitness and pathogenic potential of these strains.

Iron is an essential nutrient for microbial growth. The opportunistic pathogen Pseudomonas aeruginosa can survive under diverse conditions, including iron-depleted environments with the aid of small non-coding RNAs (sRNAs). P. aeruginosa also uses three quorum sensing (QS) systems Las, Rhl, and Pqs to coordinate virulence and infection establishment at the population level. This study links the sRNA Lrs1, which is located within the promoter of the Pqs biosynthetic operon pqsABCDE, to iron uptake regulation in the P. aeruginosa strain PAO1-L. Transcriptomics and phenotypic assays indicate that Lrs1 downregulates the production of the siderophore pyochelin but not pyoverdine, and that lrs1 regulation itself is dependent on iron availability. Although Lrs1 has been implicated in a positive feedback loop with the transcriptional regulator LasR in the strain PA14, the present findings indicate that this is not the case in PAO1-L in the tested conditions. Transcription of Lrs1 is dependent on quorum sensing, predominantly on RhlR with an auxiliary effect by PqsE. Furthermore, the Pqs system and phenazine production are modulated by Lrs1 only under iron limitation. This study identifies Lrs1 as a new QS-dependent post-transcriptional regulator of iron uptake and virulence highlighting its importance in environmental adaptation in P. aeruginosa. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/627364v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@1585ca5org.highwire.dtl.DTLVardef@2a7d55org.highwire.dtl.DTLVardef@18a712eorg.highwire.dtl.DTLVardef@c84d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Small proteins shorter than 50 amino acids have been long overlooked. A number of small proteins have been identified in several model bacteria using experimental approaches and assigned important functions in diverse cellular processes. The recent development of ribosome profiling technologies has allowed a genome-wide identification of small proteins and small ORFs (smORFs), but our incomplete understanding of small proteins hinders de novo computational prediction of smORFs in non-model bacterial species. Here, we have identified several sequence features for smORFs by a systematic analysis of all the known small proteins in E. coli, among which the translation initiation rate is the strongest determinant. By integrating these features into a support vector machine learning model, we have developed a novel sPepFinder algorithm that can predict conserved smORFs in bacterial genomes with a high accuracy of 92.8%. De novo prediction in E. coli has revealed several novel smORFs with evidence of translation supported by ribosome profiling. Further application of sPepFinder in 549 bacterial species has led to the identification of > 100,000 novel smORFs, many of which are conserved at the amino acid and nucleotide levels under purifying selection. Overall, we have established sPepFinder as a valuable tool to identify novel smORFs in both model and non-model bacterial organisms, and provided a large resource of small proteins for functional characterizations.

The worldwide rise in the prevalence of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli is a major public health concern. In Europe, ESBL carriage frequency increased then stabilized at about 6-8 %. Past antibiotic use and travel in countries with high ESBL frequency, notably South-East Asia, have repeatedly been identified as risk factors of ESBL carriage. Yet, the relative contributions of these mechanisms to the observed maintenance of a stable low frequency of ESBL in Europe remains unknown. Here, we used comprehensive data on the risk factors for carriage of ESBL-producing E. coli in the French community, alongside detailed microbiological characterization of both resistant and overall E. coli, to develop a biologically plausible mathematical model of ESBL resistance spread in France. The model also includes several mechanisms previously showed to favor coexistence such as population structure, variability in carriage duration and within-host dynamics. The level of resistance in the community implies resistant strains transmit 14% less than sensitive (95% credible interval 0.6-38%), and are cleared at a +23% larger rate (0.9-62%). ESBL resistance is predicted to be strongly associated with factors prolonging residence in the gut. Both the rate of antibiotic treatment and transmission strongly impact the frequency of ESBL in the community. In contrast, travel has little impact on ESBL frequency. Whether reducing treatment or transmission is best to reduce resistance depends on community-specific parameters. Our study opens perspectives for the quantitative study of resistance evolution and argues for future work to improve the characterization of the duration of carriage of commensal bacterial strains.

Type III CRISPR-Cas immune systems that recognize and cleave extrachromosomal RNA when active, are particularly widespread in archaea. Mechanistically, these systems have the potential to regulate gene expression of host genes on a post-transcriptional level, but very little is known about any potential accessory roles of type III-B systems beyond immunity. We have created knockout mutants of a type III-B CRISPR-Cas complex in the thermoacidophilic archaeon Saccharolobus solfataricus to investigate potential secondary functions of the type III-B system. Deletion mutants exhibited an accelerate growth but were less quickly adaptable to changes in carbon sources in their growth media. In line with this phenotype, upregulated genes were significantly enriched in functional categories of energy production and conversion, as well as with carbohydrate or amino acid transport and metabolism in RNAseq studies. Generally, a significant accumulation of genes encoding transmembrane proteins in the upregulated proportion of the transcriptome suggests interconnections between the type III-B CRISPR-Cas system and various membrane-associated processes. Notably, the deletion mutants did not lose their general virus- or plasmid defense activities indicating that this particular system might have been partially adopted for cellular regulatory roles.

AUTHOR SUMMARYThe host-pathogen interface is a biological niche in which two entities competes for essential resources. The hosts nutritional immunity restrict access to metals, while a successful pathogen overcomes these restrictions using dedicated uptake pathways. Pseudopaline is a high-affinity metallophore allowing Pseudomonas aeruginosa to acquire zinc in chelated environments. We demonstrate that this pathway is the last-resort solution to acquire zinc for this dreadful pathogen. The capacity to provide this metal to zinc-metalloproteins drives clinically relevant phenotypes, such as the capacity to form a mature and antibiotic-tolerant biofilm, or to affect the outcome of an infection. These results place pseudopaline as a potential drug target for blocking P. aeruginosa pathogenic capacity and resensitizing established biofilm to classic antibiotic treatment. ABSTRACTBiological metals are essential trace elements which are required by metalloproteins, involved in virtually every cellular, structural and catalytic function of the bacterial cell. Bacterial pathogenesis involves a tug-of-war between the host nutritional immunity, sequestering essential metals and the invading pathogens that deploy high-metal affinity uptake strategies in order to overcome these defence mechanisms. Metallophores are high-affinity, low-molecular mass metal chelators produced and secreted by bacteria to access chelated metals from the environment. Pseudopaline is a metallophore produced and secreted by Pseudomonas aeruginosa to acquire zinc when the bioavailability of this metal is severely restricted, as in the presence of a strong metal chelator such as EDTA, or during infections when the nutritional immunity of the host is active, in mammals through the production of the zing binding protein calprotectin. We show that under the conditions of metal deprivation, a pseudopaline-deficient P. aeruginosa strain exhibit a severe intracellular zinc deficiency, establishing that the pseudopaline pathway is the last-resort and unique pathway for the bacteria to acquire zinc under these restricted growth conditions. The present study explores the pleiotropic role of pseudopaline-mediated zinc acquisition on several clinically relevant phenotypes and its capacity to drive infection outcomes, placing this machinery as a promising therapeutic target for P. aeruginosas infection, acting synergistically as a pathogenicity determinant as well as an adaptative trait allowing the establishment of a mature and antibiotic resistance biofilm necessary for recalcitrant chronic infections.

1.In 2024, the World Health Organisation (WHO) classified Klebsiella pneumoniae as a maximum priority pathogen for the development of new alternatives to antibiotics. In this context, understanding the regulation of key virulence mechanisms is essential. Here, we investigated the role of the orphan quorum-sensing receptor SdiA in modulating virulence-associated processes during macrophage infection. Deletion of sdiA ({Delta}sdiA) significantly increased susceptibility to phagocytosis, as demonstrated using an amoeba predation model in which mutant strains formed larger clearance zones compared to wild-type bacteria. This phenotype was also observed in murine macrophages, where {Delta}sdiA strains exhibited increased adhesion (1.5 to 2.5-fold) and phagocytic uptake. Reduced uronic acid levels were also quantified in mutant strains, indirectly indicating a diminished capsule production, likely contributing to this enhanced phagocytosis. Despite enhanced uptake, {Delta}sdiA strains showed increased intracellular survival and replication rates within macrophages, leading to reduced host cell viability. This effect occurred despite loss of interbacterial killing capacity against E. coli, suggesting that enhanced intracellular fitness is not driven by classical antibacterial offensive mechanisms. Notably, mutant-infected macrophages displayed increased generation of reactive oxygen species (ROS), NF-{kappa}B expression, and pro-inflammatory cytokines (mCXCL10 and mTNF) production, indicating that macrophage defence mechanisms are not impaired during mutant infection. Overall, bacterial survival of {Delta}sdiA could result from overwhelming, rather than actively suppressing, host defences. Together, these findings identify SdiA as a negative regulator of phagocytosis and intracellular survival in K. pneumoniae and highlight a context-dependent role in virulence. This work provides new insights into the regulatory networks governing host-pathogen interactions and bacterial adaptation to the intracellular environment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/725935v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1d45bfdorg.highwire.dtl.DTLVardef@e3547forg.highwire.dtl.DTLVardef@c078f9org.highwire.dtl.DTLVardef@46408a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Loss of sdiA strongly affects phagocytosis, as mutant strains showed increasing adhesion (1.5 to 2.5-fold) and phagocytic uptake. Diminished capsule production could be contributing to this enhanced phagocytosis, as reduced uronic acid levels were also quantified in mutant strains. Despite being internalized at higher rates, mutants exhibited enhanced intracellular survival and replication, reducing macrophage viability. This fitness advantage occurred independently of classical offensive mechanisms, as evidenced by a lost ability to kill E. coli. Notably, mutant-infected macrophages mounted a stronger immune response, marked by elevated ROS, NF-{kappa}B expression, and pro-inflammatory cytokines production (mCXCL10 and mTNF). Together, these findings suggest that strains survive by overwhelming, rather than suppressing, host immune defences. Created with Biorender (https://www.biorender.com/). C_FIG HighlightsO_LISdiA deletion in K. pneumoniae increases susceptibility to phagocytosis. C_LIO_LIThe mutant strains exhibit reduced uronic acid levels, indicative of capsule production. C_LIO_LISdiA mutants show enhanced intracellular survival and higher macrophage death. C_LIO_LIMutant infected macrophages have higher NF-{kappa}B, TNF, and CXCL10 responses. C_LIO_LISdiA-deficient strains lose predatory capacity against E. coli. C_LI