Biology subjects

Dehio, C.

Publications and source records attributed to Dehio, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Competition against the intestinal microbiota selects for pathoadaptive traits in ESBL E. coli

The intestinal tract is a reservoir for Extended-Spectrum {beta}-Lactamase (ESBL)-producing Escherichia coli. Asymptomatic gut colonization by these pathobionts represents a major risk for extraintestinal infections. Despite clinical relevance, the genetic basis of gut colonization by ESBL E. coli remains poorly understood. Here, we determined how the microbiota shapes the fitness landscape of diverse ESBL E. coli strains, defining the functional requirements for intestinal colonization. In microbiota-depleted hosts, colonization relies mostly on metabolic functions. In contrast, in mice harbouring a microbiota, pathoadaptive functions associated with adhesion and biofilm formation are dominant determinants of E. coli fitness, together with accessory virulence functions. Consistent with these observations, experimental evolution in mice reveals convergent adaptation of ESBL E. coli to the presence of a complex microbiota through enhanced adhesion. These findings establish the microbiota as a major ecological driver of pathoadaptation in antibiotic-resistant pathobionts.

microbiology↗

bioRxiv2026

Monitoring intracellular antibiotic concentrations in real-time using allosteric biosensors

Antibiotic treatment can fail due to insufficient drug availability at the site of infection or limited accumulation within bacterial pathogens. However, it is poorly understood how antibiotics penetrate infected tissues and complex bacterial aggregates, limiting insights into the mechanisms of treatment failure. Here, we present genetically-encoded allosteric biosensors for two antibiotic classes, trimethoprim and tetracycline, which enable real-time monitoring of antibiotic concentrations inside bacterial cells. The biosensors consist of circularly permuted EGFP linked to the sensory domains DHFR or TetR. To extend this approach to low oxygen environments, we engineered an oxygen-independent trimethoprim biosensor by fusing DHFR to a circularly permuted version of the fluorogenic protein FAST. Using these biosensors, we monitored the antibiotic exposure dynamics of intracellular Salmonella enterica during macrophage infection at the single-cell level, and antibiotic penetration into anaerobic regions of Vibrio cholerae biofilms, as well as antibiotic availability in microoxic conditions in a human bladder tissue model infected with uropathogenic Escherichia coli. These fluorescent biosensors have the potential to be broadly applied for determining antibiotic distributions at infection sites with high spatial and temporal resolution.

microbiology↗

bioRxiv2026

Decoding E. coli's Gut Survival Strategies: A CRISPRi Approach Across Diets, Inflammatory Environment and Strains

Escherichia coli, a ubiquitous member of the mammalian gut microbiota, exhibits remarkable genetic diversity underpinning its commensal or pathogenic lifestyles. Deciphering the precise genetic determinants enabling E. colis adaptation within the complex and dynamic intestinal environment is critical for understanding host-microbe symbiosis and enteric disease pathogenesis. Here, we establish an in vivo CRISPR interference (CRISPRi) platform that leverages bacterial gene fitness profiles as a high-resolution functional reporter to define the molecular niche and selective forces encountered by E. coli within mice harboring a defined minimal microbial community (OligoMM12). Our investigation revealed that dietary regimens profoundly reshape E. colis metabolic landscape and that the profile of essential genes help identify cross-feeding interactions. Comparative screens across a laboratory strain (MG1655), a Uropathogenic, and Adherent-Invasive E. coli (AIEC), identify distinct genetic requirements for intestinal colonization, highlighting divergent motility, stress response, and respiration strategies. In a host inflammatory environment, we find that the AIEC strain LF82 alters its colonization pattern, shifting towards the small intestine, and adapts to the inflammatory environment by remodeling its metabolism and stress responses. Notably, we uncover a critical role for mobile genetic elements, with the observation that inflammation triggers the induction of the Gally prophage which is beneficial for fitness in the healthy gut but becomes detrimental during inflammation. These findings provide a high-resolution genetic atlas of E. colis functional adaptation and demonstrate the utility of functional genomics to probe the gut environment itself.

microbiology↗

bioRxiv2025

Perfusable 3D human urothelial model for real-time analysis of bacterial infection dynamics and therapeutic interventions

Urinary tract infections (UTIs) remain a major health burden, yet mechanistic studies are limited by the lack of experimental models that enable high spatiotemporal resolution tracking of infection dynamics, while recapitulating the stratified architecture of the bladder epithelium, urine tolerance and fluid dynamics. Here, we present a modular microphysiological platform integrating a fully stratified, urine-tolerant human urothelium cultured on standard transwell inserts within a custom-designed perfusion device compatible with live imaging. Urine flow enables real-time, high-resolution imaging of uropathogenic Escherichia coli (UPEC) infections under physiologically relevant conditions, including clearance of planktonic bacteria and nutrients replenishment, while retaining tissue-associated populations. This system revealed UPEC attachment via the type 1 fimbrial adhesin FimH and its inhibition by D-mannose treatment. Moreover, the platform captured L-form formation upon treatment with the frontline antibiotic fosfomycin and regrowth of walled bacteria following drug withdrawal. The platform further uncovered strain-specific lysis through bacteriophages in contrast to the activity of broad-spectrum antibiotics. In summary, this system constitutes a scalable platform with high predictive power for studying UTI pathogenesis and preclinical therapeutic testing.

bioengineering↗

bioRxiv2025

Genome-scale CRISPRi profiling reveals metabolic vulnerabilities of uropathogenic Escherichia coli in human urine

Urinary tract infections (UTIs) are among the most common infectious diseases, causing over 400 million cases and 260,000 deaths annually. Women are disproportionately affected, with [~]50% experiencing at least one UTI during their lifetime and 20-30% suffering from recurrent infections. Uropathogenic Escherichia coli (UPEC), which accounts for [~]75% of cases, employs diverse virulence factors to persist and evade host immunity. Rising antibiotic resistance, driven by widespread antimicrobial misuse, is eroding treatment efficacy and highlights the urgent need for alternative therapeutic strategies. To uncover novel vulnerabilities under physiologically relevant conditions, we constructed a genome-wide CRISPR interference (CRISPRi) library in the UPEC reference strain E. coli CFT073 and systematically profiled gene fitness in rich media versus human urine. The screen revealed multiple pathways that are conditionally essential for UPEC growth in urine, including iron uptake, envelope maintenance, and the biosynthesis of arginine, methionine, and branched-chain amino acids. Notably, we identified acetolactate synthase (ALS) II as the sole active isoform supporting branched-chain amino acid synthesis in urine. Functional validation further demonstrated its druggability: introducing a re-sensitizing mutation overcame the proteins intrinsic resistance to the ALS-targeting herbicide sulfometuron methyl, restoring sensitivity. These findings establish ALS II as a promising therapeutic target against UPEC.

microbiology↗

bioRxiv2025

A subset of type 4 secretion system effectors of Brucella spp. associates to outer membrane vesicles

The establishment of a replicative niche in the hostile environment of the host presents an enormous challenge for pathogens. Intracellular pathogens such as Brucella spp., the Gram-negative causative agents of Brucellosis, must subvert diverse host functions to ensure survival and replication. One of the key adaptations to achieve this is the translocation of effector proteins into host cells via its type 4 secretion system (T4SS), a key virulence factor. But effector identification in Brucella is particularly challenging, as previously identified effectors lack a conserved translocation signal, exhibit variable requirements for translocation, and in some cases appear to be translocated in a T4SS-independent manner. Here, we demonstrate that a subset of previously described T4SS effector proteins associates with outer membrane vesicles (OMVs) in different Brucella species. Most of these effector proteins encode predicted signal peptides for periplasmic export or transmembrane domains. Among them, BspC and VceA carry functional signal peptides that direct their export into the periplasm in a Sec-dependent manner. From the periplasm these proteins are subsequently secreted into the extracellular milieu, likely via the formation and release of OMVs. Our findings provide new insights into protein secretion by Brucella, suggesting that OMVs may represent an alternative secretion pathway to the T4SS.

microbiology↗

bioRxiv2024

Transcriptional antitermination integrates the expression of loci of diverse phage origin in the chimeric Bartonella Gene Transfer Agent BaGTA

Gene Transfer Agents (GTAs) are mobile genetic elements derived from bacteriophages that mediate genome-wide horizontal gene transfer (HGT) in diverse groups of prokaryotes. BaGTA, encoded by all the pathogens of the genus Bartonella, is a chimeric GTA that evolved by the domestication of two phages. The run-off-replication module ROR of one phage is integrated with the capsid production, DNA packaging and lysis machinery Bgt of a second phage. Restricted to a self-sacrificing subset of the bacterial population, the position-specific DNA amplification and packaging of a genomic plasticity region enriched for genes involved in host interaction and adaptation selectively enhances the HGT frequency of these pathogenicity genes. This feature of BaGTA is considered a key innovation underlying the evolutionary success of Bartonella. Little is known, however, about the mechanism mediating the coordinated expression of the ror and bgt loci. Here, we established the regulatory hierarchy, with ror acting upstream of the capsid gene cluster bgtA-K. BrrG, encoded by the ror locus, controls the transcription of the bgtA-K operon by functioning as a processive antiterminator. This study provides the first insights into the mechanism controlling the coordinated expression of the two BaGTA modules of divergent phage origin. Beyond BaGTA, we propose that antitermination is a broadly relevant mechanism for controlling HGT by GTAs of the Alphaproteobacteria.

microbiology↗

bioRxiv2024

Full-length structure of the host targeted bacterial effector Bep1 reveals a novel structural domain conserved in FIC effector proteins from Bartonella

Bacterial effector proteins translocated via a type-IV secretion system (T4SS) typically harbor a C-terminal segment required for recognition by the type-IV secretion coupling protein 1. In the -proteobacterial pathogen Bartonella, the signal is bipartite being composed of a BID (Bep intracellular delivery) domain and a positively charged C-terminal tail 2. Here, we show the crystal structure of full length Bartonella effector protein 1 (Bep1), which shows a novel FIC - OB - BAS(BID) domain arrangement conserved in the majority of Beps with the BID domain inserted into the newly discovered BAS parent domain. We propose that the BAS domain is necessary for the overall "boomerang"-like shape of Bep1 and that it plays a role during translocation through the T4SS.

microbiology↗

bioRxiv2024

Translocation of YopJ family effector proteins through the VirB/VirD4 T4SS of Bartonella

The evolutionary conserved YopJ family comprises numerous type-III-secretion system (T3SS) effectors of diverse mammalian and plant pathogens that acetylate host proteins to dampen immune responses. Acetylation is mediated by a central acetyltransferase domain that is flanked by conserved regulatory sequences, while a non-conserved N-terminal extension encodes the T3SS-specific translocation signal. Bartonella spp. are facultative-intracellular pathogens causing intra-erythrocytic bacteremia in their mammalian reservoirs and diverse disease manifestations in incidentally infected humans. Bartonellae do not encode a T3SS, but most species possess a type-IV-secretion system (T4SS) to translocate Bartonella effector proteins (Beps) into host cells. Here we report that the YopJ homologs present in Bartonellae species represent genuine T4SS effectors. Like YopJ family T3SS effectors of mammalian pathogens, the Bartonella YopJ-like effector A (ByeA) of Bartonella taylorii also targets MAP kinase signaling to dampen pro-inflammatory responses, however, translocation depends on a functional T4SS. A split-NanoLuc luciferase-based translocation assay identified sequences required for T4SS-dependent translocation in conserved regulatory regions at the C-terminus and proximal to the N-terminus of ByeA. The T3SS effectors YopP from Yersinia enterocolitica and AvrA from Salmonella Typhimurium were also translocated via the Bartonella T4SS, while ByeA was not translocated via the Yersinia T3SS. Our data suggest that YopJ family T3SS effectors may have evolved from an ancestral T4SS effector, such as ByeA of Bartonella. In this evolutionary scenario, the signal for T4SS-dependent translocation encoded by N- and C-terminal sequences remained functional in the derived T3SS effectors due to the essential role these sequences coincidentally play in regulating acetyltransferase activity. Significance StatementBacterial pathogens use diverse secretion systems to translocate effector proteins into eukaryotic host cells. Evolutionary successful translocation systems and effector proteins have been acquired by many plant and animal pathogens via horizontal gene transfer. The YopJ family comprises numerous T3SS effectors that share a unique acetyltransferase activity that interferes with various host cell functions. Our study revealed that YopJ homologs in the pathogen Bartonella are genuine T4SS effectors and implies an evolutionary scenario in which T3SS-dependent YopJ family effectors may have evolved from such an ancestral T4SS effector by fusion of an N-terminal type-III-secretion signal. Such switches in secretion system specificity of host-targeted effectors may represent an underappreciated phenomenon in effector evolution.

microbiology↗

bioRxiv2024

The putative Type 4 secretion system effector BspD is involved in maintaining envelope integrity of the pathogen Brucella

Brucellosis is a debilitating disease caused by the Gram-negative, facultative intracellular zoonotic pathogen Brucella. En route to its intracellular replicative niche, Brucella encounters various stressful environments that may compromise envelop integrity. Here we show, that the proposed Type 4 secretion system (T4SS) effector BspD is a conserved protein of the Rhizobiales, which does not show signs of co-evolution with the presence of a T4SS or a certain lifestyle. We further present data indicating that BspD is critical for Brucella abortus envelope integrity in the stationary phase and in the presence of EDTA, a compound known to destabilize the outer membrane. Deletion of bspD resulted in abnormal bacterial morphologies, indicating its involvement in maintaining envelope integrity. Additionally, the absence of BspD led to the formation of fewer and smaller intracellular microcolonies in a macrophage infection model. From our observations, we propose that BspD of B. abortus is critical for preserving the integrity of the bacterial envelope, particularly under stressful conditions, which may enhance Brucellas ability to survive within host cells. ImportanceBrucellosis, caused by the intracellular pathogen Brucella, poses a significant health threat. Understanding how Brucella adapts to stressful environments is crucial. This study unveils BspD, a conserved protein within the Rhizobiales order, as a key player in maintaining Brucellas envelope integrity. Remarkably, BspDs function appears independent of the presence of a Type 4 Secretion System (T4SS) or specific lifestyle. Deletion of bspD resulted in compromised envelope integrity, abnormal bacterial morphologies, and reduced intracellular microcolony formation. These findings underscore BspDs critical role, particularly in stressful conditions like the stationary phase and EDTA exposure, and highlight its significance in Brucellas survival within host cells. This elucidation deepens our understanding of Brucella pathogenesis and may inform future therapeutic strategies against brucellosis.

microbiology↗

bioRxiv2023

Structure-function analysis of the cyclic beta-1,2-glucan synthase

The synthesis of complex sugars is a key aspect of microbial biology. Cyclic {beta}-1,2-glucan (C{beta}G) is a circular polysaccharide critical for host interactions of many bacteria, including major pathogens of humans (Brucella) and plants (Agrobacterium). C{beta}G is produced by the cyclic glucan synthase (Cgs), a massive multi-domain membrane protein. So far, its structure as well as the mechanisms underlining the synthesis have not been clarified. Here we use cryo-electron microscopy (cryo-EM) and functional approaches to study Cgs from A. tumefaciens. We were able to determine the structure of this complex protein machinery and clarify key aspects of C{beta}G synthesis. Our research opens new possibilities for combating pathogens that rely on polysaccharide virulence factors and can lead to new synthetic biology approaches for producing complex cyclic sugars.

molecular biology↗

bioRxiv2022

BNIP3L-mediated mitophagy triggered by Brucella in host cells is required for bacterial egress

The facultative intracellular pathogen Brucella abortus interacts with several organelles of the host cell to reach its replicative niche inside the endoplasmic reticulum. However, little is known about the interplay between the bacteria and the host cell mitochondria. Here, we showed that B. abortus triggers a strong mitochondrial network fragmentation accompanied by mitophagy and the formation of mitochondrial Brucella-containing vacuoles in the late steps of cellular infection. The expression of the mitophagy receptor BNIP3L induced by B. abortus is essential for these events and relies on the iron-dependent stabilization of the hypoxia-inducible factor 1 alpha. Functionally, BNIP3L-mediated mitophagy appears to be advantageous for bacterial exit of the host cell as BNIP3L depletion drastically reduced the number of reinfection events. Altogether, these findings highlight the intricate link between Brucella trafficking and the mitochondria during host cell infection.

microbiology↗

bioRxiv2022

Antibiotic persistence of Brucella abortus in its protective intracellular niche

BackgroundHuman Brucellosis caused by the facultative intracellular pathogen Brucella spp. is an endemic bacterial zoonosis manifesting as acute or chronic infections associated with high morbidity. Treatment typically involves a combination therapy of two antibiotics administered for several weeks to month, but despite this harsh treatment relapses occur at a rate of 5-15%. Although poor compliance and reinfection may account for a fraction of the observed relapse cases, it is apparent that the properties of the infectious agent itself may play a decisive role in this phenomenon. Methodology/Principal findingsWe used B. abortus carrying a dual reporter in a macrophage infection model to gain a better understanding of the efficacy of recommended therapies in cellulo. For this we used automated fluorescent microscopy as a prime read-out and developed specific CellProfiler pipelines to score infected macrophages at the population and the single cell level. Combining microscopy of constitutive and induced reporters with classical CFU determination, we quantified the protective nature of the Brucella intracellular niche to various antibiotics and the ability of B. abortus to persist in cellulo despite harsh antibiotic treatments. Conclusion/SignificanceWe demonstrate that treatment of infected macrophages with antibiotics at recommended concentrations fails to fully prevent growth and persistence of B. abortus in cellulo, which may be explained by a protective nature of the intracellular niche. Moreover, we show the presence of bona fide intracellular persisters upon antibiotic treatment, which are metabolically active and retain the full infectious potential, therefore constituting a plausible reservoir for reinfection and relapse. In conclusion, our results highlight the need to extend the spectrum of models to test new antimicrobial therapies for Brucellosis to better reflect the in vivo infection environment, and to develop therapeutic approaches targeting the persister subpopulation. Authors SummaryBrucellosis is a zoonosis endemic to many low- and middle-income countries around the world. Recommended therapies by the WHO are comprised of at least two antibiotics for several weeks, sometimes month. Despite these harsh treatments relapses are frequent. The underlying reasons for these relapses, besides reinfection and non-compliance to treatment, are unknown. Our study shows that Brucella abortus can form so called "persisters" under recommended treatments in rich broth but also inside macrophages. This small bacterial subpopulation survives antibiotic treatment and resumes growth after removal of the antibiotics and could therefore serve as a reservoir for relapses in human Brucellosis. Furthermore, we show that the macrophage intracellular niche of Brucella has protective properties against recommended antibiotics as observed for other intracellular pathogens, highlighting the necessity to develop new infection models to assess antibiotic efficacy.

microbiology↗

bioRxiv2022

Bartonella taylorii: A model organism for studying Bartonella infection in vitro and in vivo

Bartonella spp. are Gram-negative facultative intracellular pathogens that infect diverse mammals and cause a long-lasting intra-erythrocytic bacteremia in their natural host. These bacteria translocate Bartonella effector proteins (Beps) into host cells via their VirB/VirD4 type 4 secretion system (T4SS) in order to subvert host cellular functions, thereby leading to the downregulation of innate immune responses. Most studies on the functional analysis of the VirB/VirD4 T4SS and the Beps were performed with the major zoonotic pathogen Bartonella henselae for which efficient in vitro infection protocols have been established. However, its natural host, the cat, is unsuitable as an experimental infection model. In vivo studies were mostly confined to rodent models using rodent-specific Bartonella species, while the in vitro infection protocols devised for B. henselae are not transferable for those pathogens. The disparities of in vitro and in vivo studies in different species has hampered progress in our understanding of Bartonella pathogenesis. Here we describe the murine-specific strain B. taylorii IBS296 as a new model organism facilitating the study of bacterial pathogenesis both in vitro in cell cultures and in vivo in laboratory mice. We implemented the split NanoLuc luciferase-based translocation assay to study BepD translocation through the VirB/VirD4 T4SS. We found increased effector-translocation into host cells if the bacteria were grown on tryptic soy agar (TSA) plates and experienced a temperature shift immediately before infection. The improved infectivity in vitro could be correlated to an upregulation of the VirB/VirD4 T4SS. Using our adapted infection protocols, we showed BepD-dependent immunomodulatory phenotypes in vitro. In mice, the implemented growth conditions enabled infection by a massively reduced inoculum without having an impact on the course of the intra-erythrocytic bacteremia. The established model opens new avenues to study the role of the VirB/VirD4 T4SS and the translocated Bep effectors in vitro and in vivo.

microbiology↗

bioRxiv2021

The Bartonella autotransporter CFA is a protective antigen and hypervariable target of neutralizing antibodies blocking erythrocyte infection

Antibodies are key to the clearance of Bartonella bacteremia, but the mechanisms and targets of protective antibodies are unknown and bacterial evasion strategies remain elusive. We studied experimental Bartonella taylorii infection of mice, its natural host, and investigated protective immune responses. Clearance of bacteremia depended on specific antibodies that interfere with bacterial attachment to erythrocytes. Accordingly, antibodies were effective in the absence of complement and Fc-receptors. Moreover, they formed independently of B-cell hypermutation and isotype class switch. The cloning of neutralizing monoclonal antibodies (mAbs) led to the identification of the bacterial autotransporter CFA as a protective antibody target, and vaccination against CFA protected against Bartonella bacteremia. MAb binding mapped to a region of CFA that is hypervariable in both human- and mouse-pathogenic Bartonella strains, suggesting mutational antibody evasion. These insights further our understanding of Bartonella immunity and immune evasion and elucidate mechanisms driving high Bartonella prevalence in the wild.

microbiology↗

bioRxiv2020

Structural basis for selective targeting of Rac subfamily GTPases by a bacterial effector protein

SummaryRas-homology (Rho) family GTPases are conserved molecular switches controlling fundamental cellular activities in eukaryotic cells. As such, they are targeted by numerous bacterial toxins and effector proteins, which have been intensively investigated regarding their biochemical activities and discrete target spectra; however, molecular mechanisms of target selectivity have remained elusive. Here, we report a bacterial effector protein that targets all four Rac subfamily members of Rho family GTPases, but none of the closely related Cdc42 or RhoA subfamilies. This exquisite target selectivity of the FIC domain AMP-transferase Bep1 from Bartonella rochalimae is based on electrostatic interactions with a subfamily-specific pair of residues in the nucleotide-binding motif and the Rho insert helix. Residue substitutions at the identified positions in Cdc42 facilitate modification by Bep1, while corresponding Cdc42-like substitutions in Rac1 greatly diminish modification. Our study establishes a structural paradigm for target selectivity towards Rac subfamily GTPases and provides a highly selective tool for their functional analysis.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Bartonella effector protein C mediates actin stress fiber formation via recruitment of GEF-H1 to the plasma membrane

Bartonellae are Gram-negative facultative-intracellular pathogens that use a type-IV-secretion system (T4SS) to translocate a cocktail of Bartonella effector proteins (Beps) into host cells to modulate diverse cellular functions. BepC was initially reported to act in concert with BepF in triggering major actin cytoskeletal rearrangements that result in the internalization of a large bacterial aggregate by the so-called invasome. Later, infection studies with bepC deletion mutants and ectopic expression of BepC have implicated this effector in triggering an actin-dependent cell contractility phenotype characterized by fragmentation of migrating cells due to deficient rear detachment at the trailing edge, and BepE was shown to counterbalance this remarkable phenotype. However, the molecular mechanism of how BepC triggers cytoskeletal changes and the host factors involved remained elusive. Using infection assays, we show here that T4SS-mediated transfer of BepC is sufficient to trigger stress fiber formation in non-migrating epithelial cells and additionally cell fragmentation in migrating endothelial cells. Interactomic analysis revealed binding of BepC to a complex of the Rho guanine nucleotide exchange factor GEF-H1 and the serine/threonine-protein kinase MRCK. Knock-out cell lines revealed that only GEF-H1 is required for mediating BepC-triggered stress fiber formation and inhibitor studies implicated activation of the RhoA/ROCK pathway downstream of GEF-H1. Ectopic co-expression of tagged versions of GEF-H1 and BepC truncations revealed that the C-terminal Bep intracellular delivery (BID) domain facilitated anchorage of BepC to the plasma membrane, whereas the N-terminal filamentation induced by cAMP (FIC) domain facilitated binding of GEF-H1. While FIC domains typically mediate post-translational modifications, most prominently AMPylation, a mutant with quadruple amino acid exchanges in the putative active site indicated that the BepC FIC domain acts in a non-catalytic manner to activate GEF-H1. Our data support a model in which BepC activates the RhoA/ROCK pathway by re-localization of GEF-H1 from microtubules to the plasma membrane. Author SummaryA wide variety of bacterial pathogens evolved numerous virulence factors to subvert cellular processes in support of a successful infection process. Likewise, bacteria of the genus Bartonella translocate a cocktail of effector proteins (Beps) via a type-IV-secretion system into infected cells in order to interfere with host signaling processes involved in cytoskeletal dynamics, apoptosis control, and innate immune responses. In this study, we demonstrate that BepC triggers actin stress fiber formation and a linked cell fragmentation phenotype resulting from distortion of rear-end retraction during cell migration. The ability of BepC to induce actin stress fiber formation is directly associated with its ability to bind GEF-H1, an activator of the RhoA pathway that is sequestered in an inactive state when bound to microtubules, but becomes activated upon release to the cytoplasm. Our findings suggest that BepC is anchored via its BID domain to the plasma membrane where it recruits GEF-H1 via its FIC domain, eventually activating the RhoA/ROCK signaling pathway and leading to stress fiber formation.

microbiology↗

bioRxiv2018

A role for VPS35 in Brucella intracellular replication revealed by genome-wide siRNA screening

Brucella, the causing agent of brucellosis, is a major zoonotic pathogen with worldwide distribution. Brucella resides and replicates inside infected host cells in membrane-bound compartments called BCVs (Brucella-containing vacuoles). Following uptake, Brucella resides in eBCVs (endosomal BCVs) that gradually mature from early to late endosomal features. Through a poorly understood process that is key to the intracellular lifestyle of Brucella, the eBCV escapes fusion with lysosomes by transitioning to the rBCV (replicative BCV), a replicative niche directly connected to the endoplasmic reticulum (ER). Despite the notion that this complex intracellular lifestyle must depend on a multitude of host factors, a holistic view on which of these components control Brucella cell entry, trafficking and replication is still missing. Here we used a systematic cell-based siRNA knockdown screen in HeLa cells infected with Brucella abortus and identified 425 components of the human infectome for Brucella infection. These include multiple components of pathways involved in central processes such as cell cycle, actin cytoskeleton dynamics or vesicular trafficking. Using assays for pathogen entry, knockdown complementation and co-localization at single-cell resolution, we identified the requirement of the VPS retromer for Brucella to escape the lysosomal degradative pathway and to establish its intracellular replicative niche. We thus validated a component of the VPS retromer as novel host factor critical for Brucella intracellular trafficking. Further, our genome-wide data shed light on the interplay between central host processes and the biogenesis of the Brucella replicative niche.\n\nImportanceWith >300,000 new cases of human brucellosis annually, Brucella is regarded as one of the most important zoonotic bacterial pathogen worldwide. The causing agent of brucellosis resides inside host cells within vacuoles termed Brucella containing vacuoles (BCVs). Although few host components required to escape the degradative lysosomal pathway and to establish the ER-derived replicative BCV (rBCV) have already been identified, the global understanding of this highly coordinated process is still partial and many factors remain unknown. To gain a deeper insight into these fundamental questions we performed a genome-wide RNA interference (RNAi) screen aiming at discovering novel host factors involved in the Brucella intracellular cycle. We identified 425 host proteins that contribute to Brucella cellular entry, intracellular trafficking, and replication. Together, this study sheds light on previously unknown host pathways required for the Brucella infection cycle and highlights the VPS retromer components as critical factors for the establishment of the Brucella intracellular replicative niche.

microbiology↗