Biology subjects

Blondel, C. J.

Publications and source records attributed to Blondel, C. J..

7 recordsLinked to original sources

bioRxiv2026

VopU is a novel T3SS effector protein with mono-ADP-ribosyltransferase activity and a non-canonical H-Y-Q catalytic triad

Vibrio parahaemolyticus utilizes its second Type III Secretion System (T3SS2) to deliver a suite of effector proteins that subvert eukaryotic host cell processes. In this study, we characterize VPA1312, renamed VopU, as a novel T3SS2 effector. We demonstrate that VopU is translocated into infected cells via the T3SS2 and independently of the VocC chaperone. From bioinformatic analyses, VopU is a member of a family of proteins distributed across diverse bacterial taxa and in some cases associated with T6SS gene clusters and plasmids. Sequence, structure-based, and functional analysis reveals that VopU harbors an ADP-ribosyltransferase domain with a non-canonical H-Y-Q catalytic triad, a motif not previously described in naturally occurring mono-ADP-ribosyltransferases. Heterologous expression of VopU within transfected cells or translocation of VopU during infection results in the ADP-ribosylation of a small protein of approximately 18 kDa protein distinct from the target of the previously described ADP-ribosyltransferase effector, VopT. While VopU is not essential for T3SS2-dependent cytotoxicity or intracellular survival of V. parahaemolyticus, heterologous expression of VopU induces host cell rounding and a transcriptional host stress response, likely linked to NAD depletion. These findings provide the initial characterization of this novel family of mono-ADP-ribosyltransferases and expand the known repertoire of bacterial effectors that mediate orthogonal post-translational modifications during host infection. IMPORTANCEVibrio parahaemolyticus is a leading cause of seafood-borne illness worldwide. To cause disease, the bacterium relies on a Type III Secretion System to deliver effector proteins into the cytosol of infected cells, subverting cellular processes. In this study, we identified a novel effector protein, VopU, which performs a post-translational modification known as ADP-ribosylation using a non-canonical catalytic triad (H-Y-Q). This is the first description of a naturally occurring mono-ART with this specific motif. We showed that VopU homologs are distributed across other bacterial species, and that VopU is active during infection of host cells. Together, our findings identify a novel family of effectors that can modify host proteins and potentially contribute to host colonization during infection.

microbiology↗

bioRxiv2023

Identification and distribution of novel candidate T6SS effectors encoded in Salmonella Pathogenicity Island 6

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.

microbiology↗

bioRxiv2022

The Vibrio Type III Secretion System 2 is not restricted to the Vibrionaceae and encodes differentially distributed repertoires of effector proteins

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis worldwide. A distinctive feature of the O3:K6 pandemic clone, and its derivatives, is the presence of a second, phylogenetically distinct, Type III Secretion System (T3SS2) encoded within the genomic island VPaI-7. The T3SS2 allows the delivery of effector proteins directly into the cytosol of infected eukaryotic cells to subvert key host cell processes, critical for V. parahaemolyticus to colonize and cause disease. Furthermore, the T3SS2 also increases the environmental fitness of V. parahaemolyticus in its interaction with bacterivorous protists; hence it has been proposed that it contributed to the global oceanic spread of the pandemic clone. Several reports have identified T3SS2-related genes in Vibrio and non-Vibrio species, suggesting that the T3SS2 gene cluster is not restricted to the Vibrionaceae and can mobilize through horizontal gene transfer events. In this work, we performed a large-scale genomic analysis to determine the phylogenetic distribution of the T3SS2 gene cluster and its repertoire of effector proteins. We identified putative T3SS2 gene clusters in 1130 bacterial genomes from 8 bacterial genera, 5 bacterial families and 47 bacterial species. A hierarchical clustering analysis allowed us to define 6 T3SS2 subgroups (I-VI) with different repertoires of effector proteins, redefining the concepts of T3SS2 core and accessory effector proteins. Finally, we identified a subset of T3SS2 gene clusters (subgroup VI) that lack most T3SS2 effector proteins described to date and provided a list of 10 novel effector candidates for this subgroup through bioinformatic analysis. Collectively, our findings indicate that the T3SS2 extends beyond the Vibrionaceae family and suggest that different effector protein repertories could have a differential impact on the pathogenic potential and environmental fitness of each bacteria that have acquired the Vibrio T3SS2 gene cluster. DATA SUMMARYO_LIAll genome sequences used in this study were downloaded from the National Center for Biotechnology Information (NCBI) RefSeq or GenBank databases (See Table S1 for accession numbers). C_LIO_LIFiles for the T3SS2 reconstructed phylogenetic tree (Newick tree and MSA fasta file), hierarchical clustering data analysis file from MORPHEUS, Table S1 with genome accession numbers and all the data of the absence/presence of T3SS2-related components, and Table S2 with the prediction of novel effector proteins are available as part of the online Supporting Dataset at the Zenodo data repository (https://doi.org/10.5281/zenodo.7016552). The T3SS2 phylogenetic tree can be interactively visualized in https://itol.embl.de/tree/19016190125374711626959067# C_LI

microbiology↗

bioRxiv2020

Identification of a family of Vibrio type III secretion system effectors that contain a conserved serine/threonine kinase domain

Vibrio parahaemolyticus is a marine Gram-negative bacterium that is a leading cause of seafood-borne gastroenteritis. Pandemic strains of V. parahaemolyticus rely on a specialized protein secretion machinery known as the type III secretion system 2 (T3SS2) to cause disease. The T3SS2 mediates the delivery of effector proteins into the cytosol of infected cells, where they subvert multiple cellular pathways. Here, we identify a new T3SS2 effector protein encoded by VPA1328 (VP_RS21530) in V. parahaemolyticus RIMD2210633. Bioinformatic analysis revealed that VPA1328 is part of a larger family of uncharacterized T3SS effector proteins with homology to the VopG effector protein in V. cholerae AM-19226. These VopG-like proteins are found in many but not all T3SS2 gene clusters and are distributed among diverse Vibrio species including V. parahaemolyticus, V. cholerae, V. mimicus, and V. diabolicus and also in Shewanella baltica. Structure-based prediction analyses uncovered the presence of a conserved C-terminal kinase domain in VopG orthologs, similar to the serine/threonine kinase domain found in the NleH family of T3SS effector proteins. However, in contrast to NleH effector proteins, in tissue culture-based infections, VopG did not impede host cell death or suppress IL-8 secretion, suggesting a yet undefined role for VopG during V. parahaemolyticus infection. Collectively, our work reveals that VopG effector proteins, a new family of likely serine/threonine kinases, is widely distributed in the T3SS2 effector armamentarium among marine bacteria. IMPORTANCEVibrio parahaemolyticus is the leading bacterial cause of seafood-borne gastroenteritis worldwide. The pathogen relies on a type III secretion system to deliver a variety of effector proteins into the cytosol of infected cells to subvert cellular function. In this study, we identified a novel Vibrio parahaemolyticus effector protein that is similar to the VopG effector of Vibrio cholerae. VopG-like effectors were found in diverse Vibrio species and contain a conserved serine/threonine kinase domain that bears similarity to the kinase domain in the EHEC and Shigella NleH effectors that manipulate host cell survival pathways and host immune responses. Together our findings identify a new family of Vibrio effector proteins and highlight the role of horizontal gene transfer events among marine bacteria in shaping T3SS gene clusters.

microbiology↗

bioRxiv2020

Genomic analysis of the diversity, antimicrobial resistance and virulence potential of Campylobacter jejuni and Campylobacter coli strains from a private health care center in Central Chile

Campylobacter jejuni and Campylobacter coli are the leading cause of human gastroenteritis in the industrialized world and an emerging threat in developing countries. The incidence of campylobacteriosis in South America is greatly underestimated, mostly due to the lack of adequate diagnostic methods. Accordingly, there is limited genomic and epidemiological data from this region. In the present study, we performed a genome-wide analysis of the genetic diversity, virulence, and antimicrobial resistance of the largest collection of clinical C. jejuni and C. coli strains from Chile available to date (n=81), collected in 2017-2019 in Santiago, Chile. This culture collection accounts for over a third of the genome sequences available of clinical strains from South America. cgMLST analysis identified high genetic diversity as well as 13 novel STs and alleles in both C. jejuni and C. coli. Pangenome and virulome analyses showed a differential distribution of virulence factors, including both plasmid and chromosomally encoded T6SSs and T4SSs. Resistome analysis predicted widespread resistance to fluoroquinolones, but low rates of erythromycin resistance. This study provides valuable genomic and epidemiological data and highlights the need for further genomic epidemiology studies in Chile and other South American countries to better understand molecular epidemiology and antimicrobial resistance of this emerging intestinal pathogen. AUTHOR SUMMARYCampylobacter is the leading cause of bacterial gastroenteritis worldwide and an emerging and neglected pathogen in South America. In this study, we performed an in-depth analysis of the genome sequences of 69 C. jejuni and 12 C. coli clinical strains isolated from Chile, which account for over a third of the sequences from clinical strains available from South America. We identified a high genetic diversity among C. jejuni strains and the unexpected identification of clade 3 C. coli strains, which are infrequently isolated from humans in other regions of the world. Most strains harbored the virulence factors described for Campylobacter. While [~]40% of strains harbored mutation in the gyrA gene described to confer fluoroquinolone resistance, very few strains encoded the determinants linked to macrolide resistance, currently used for the treatment of campylobacteriosis. Our study contributes to our knowledge of this important foodborne pathogen providing valuable data from South America.

microbiology↗

bioRxiv2019

Transposon-insertion sequencing screens unveil requirements for EHEC growth and intestinal colonization

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an important food-borne pathogen that colonizes the colon. Transposon-insertion sequencing (TIS) was used to identify genes required for EHEC and commensal E. coli K-12 growth in vitro and for EHEC growth in vivo in the infant rabbit colon. Surprisingly, many conserved loci contribute to EHECs but not to K-12s growth in vitro, suggesting that gene acquisition during EHEC evolution has heightened the pathogens reliance on certain metabolic processes that are dispensable for K-12. There was a restrictive bottleneck for EHEC colonization of the rabbit colon, which complicated identification of EHEC genes facilitating growth in vivo. Both a refined version of an existing analytic framework as well as PCA-based analysis were used to compensate for the effects of the infection bottleneck. These analyses confirmed that the EHEC LEE-encoded type III secretion apparatus is required for growth in vivo and revealed that only a few effectors are critical for in vivo fitness. Numerous mutants not previously associated with EHEC survival/growth in vivo also appeared attenuated in vivo, and a subset of these putative in vivo fitness factors were validated. Some were found to contribute to efficient type-three secretion while others, including tatABC, oxyR, envC, acrAB, and cvpA, promote EHEC resistance to host-derived stresses encountered in vivo. cvpA, which is also required for intestinal growth of several other enteric pathogens, proved to be required for EHEC, Vibrio cholerae and Vibrio parahaemolyticus resistance to the bile salt deoxycholate. Collectively, our findings provide a comprehensive framework for understanding EHEC growth in the intestine. Author SummaryEnterohemorrhagic E. coli (EHEC) are important food-borne pathogens that infect the colon. We created a highly saturated EHEC transposon library and used transposon insertion sequencing to identify the genes required for EHEC growth in vitro and in vivo in the infant rabbit colon. We found that there is a large infection bottleneck in the rabbit model of intestinal colonization, and refined two analytic approaches to facilitate rigorous identification of new EHEC genes that promote fitness in vivo. Besides the known type III secretion system, more than 200 additional genes were found to contribute to EHEC survival and/or growth within the intestine. The requirement for some of these new in vivo fitness factors was confirmed, and their contributions to infection were investigated. This set of genes should be of considerable value for future studies elucidating the processes that enable the pathogen to proliferate in vivo and for design of new therapeutics.

microbiology↗

bioRxiv2018

CRISPR screen reveals that EHEC’s T3SS and Shiga toxin rely on shared host factors for infection

Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors - a type III secretion system (T3SS) and Shiga toxins (Stx) - that are required for the pathogen to colonize the intestine and cause diarrheal disease. Here, we carried out a genome-wide CRISPR/Cas9 loss-of-function screen to identify host loci that facilitate EHEC infection of intestinal epithelial cells. Many of the guide RNAs identified targeted loci known to be associated with sphingolipid biosynthesis, particularly for production of globotriaosylceramide (Gb3), the Stx receptor. Two loci (TM9SF2 and LAPTM4A) with largely unknown functions were also targeted. Mutations in these loci not only rescued cells from Stx-mediated cell death, but also prevented cytotoxicity associated with the EHEC T3SS. These mutations interfered with early events associated with T3SS and Stx pathogenicity, markedly reducing entry of T3SS effectors into host cells and binding of Stx. The convergence of Stx and T3SS onto overlapping host targets provides guidance for design of new host-directed therapeutic agents to counter EHEC infection.\n\nImportanceEnterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors - a type III secretion system (T3SS) and Shiga toxins (Stx) - that are required for colonizing the intestine and causing diarrheal disease. We screened a genome-wide collection of CRISPR mutants derived from intestinal epithelial cells and identified mutants with enhanced survival following EHEC infection. Many had mutations that disrupted synthesis of a subset of lipids (sphingolipids) that includes the Stx receptor globotriaosylceramide (Gb3), and hence protect against Stx intoxication. Unexpectedly, we found that sphingolipids also mediate early events associated with T3SS pathogenicity. Since antibiotics are contraindicated for the treatment of EHEC, therapeutics targeting sphingolipid biosynthesis are a promising alternative, as they could provide protection against both of the pathogens key virulence factors.

microbiology↗