Contact-dependent Growth Inhibition Research Articles

Biocontrol of Escherichia coli O157:H7 by Enterobacter asburiae AEB30 on intact cantaloupe melons.

Escherichia coli O157:H7 causes >73,000 foodborne illnesses in the United States annually, many of which have been associated with fresh ready-to-eat produce including cantaloupe melons. In this study, we created a produce-associated bacterial (PAB) library containing >7500 isolates and screened them for the ability to inhibit the growth of E. coli O157:H7 using an invitro fluorescence-based growth assay. One isolate, identified by 16S and whole-genome sequence analysis as Enterobacter asburiae, was able to inhibit the growth of E. coli by ~30-fold invitro and produced zones of inhibition between 13 and 21 mm against 12 E. coli outbreak strains in an agar spot assay. We demonstrated that E. asburiae AEB30 was able to grow, persist and inhibit the growth of E. coli on cantaloupe melons under simulated pre- and post-harvest conditions. Analysis of the E. asburiae AEB30 genome revealed an operon encoding a contact-dependent growth inhibition (CDI) system that when mutated resulted in the loss of E. coli growth inhibition. These data suggest that E. asburiae AEB30 is a potential biocontrol agent to prevent E. coli contamination of cantaloupe melons in both pre- and post-harvest environments and that its mode of action is via a CDI system.

The Love and Hate Relationship between T5SS and Other Secretion Systems in Bacteria.

Bacteria have existed on Earth for billions of years, exhibiting ubiquity and involvement in various biological activities. To ensure survival, bacteria usually release and secrete effector proteins to acquire nutrients and compete with other microorganisms for living space during long-term evolution. Consequently, bacteria have developed a range of secretion systems, which are complex macromolecular transport machines responsible for transporting proteins across the bacterial cell membranes. Among them, one particular secretion system that stands out from the rest is the type V secretion system (T5SS), known as the "autotransporter". Bacterial activities mediated by T5SS include adherence to host cells or the extracellular matrix, invasion of host cells, immune evasion and serum resistance, contact-dependent growth inhibition, cytotoxicity, intracellular flow, protease activity, autoaggregation, and biofilm formation. In a bacterial body, it is not enough to rely on T5SS alone; in most cases, T5SS cooperates with other secretion systems to carry out bacterial life activities, but regardless of how good the relationship is, there is friction between the secretion systems. T5SS and T1SS/T2SS/T3SS/T6SS all play a synergistic role in the pathogenic processes of bacteria, such as nutrient acquisition, pathogenicity enhancement, and immune modulation, but T5SS indirectly inhibits the function of T4SS. This could be considered a love-hate relationship between secretion systems. This paper uses the systematic literature review methodology to review 117 journal articles published within the period from 1995 to 2024, which are all available from the PubMed, Web of Science, and Scopus databases and aim to elucidate the link between T5SS and other secretion systems, providing clues for future prevention and control of bacterial diseases.

Relaxed specificity of BcpB transporters mediates interactions between Burkholderia cepacia complex contact-dependent growth inhibition systems.

Belonging to the two-partner secretion family of proteins, contact-dependent growth inhibition (CDI) systems mediate interbacterial antagonism among closely related Gram-negative bacteria. The toxic portion of a large surface protein, BcpA/CdiA, is delivered to the cytoplasm of neighboring cells where it inhibits growth. Translocation of the antibacterial polypeptide out of the producing cell requires an associated outer membrane transporter, BcpB/CdiB. Some bacteria, including many Burkholderia species, encode multiple distinct CDI systems, but whether there is interaction between these systems is largely unknown. Using Burkholderia cepacia complex species as a model, here we show that related BcpB transporters exhibit considerable secretion flexibility and can secrete both cognate and non-cognate BcpA substrates. We also identified an additional unique Burkholderia dolosa CDI system capable of mediating interbacterial competition and demonstrated that its BcpB transporter has similar relaxed substrate specificity. Our results showed that two BcpB transporters (BcpB-2 and BcpB-3) were able to secrete all four of the B. dolosa BcpA toxins, while one transporter (BcpB-1) appeared unable to secrete even its cognate BcpA substrate under the tested conditions. This flexibility provided a competitive advantage, as strains lacking the full repertoire of BcpB proteins had decreased CDI activity. Similar results were obtained in Burkholderia multivorans, suggesting that secretion flexibility may be a conserved feature of Burkholderia CDI systems. Together these findings suggest that the interaction between distinct CDI systems enhances the efficiency of bacterial antagonism. IMPORTANCE The Burkholderia cepacia complex (Bcc) is a group of related opportunistic bacterial pathogens that occupy a diverse range of ecological niches and exacerbate disease in patients with underlying conditions. Contact-dependent growth inhibition (CDI) system proteins, produced by Gram-negative bacteria, contain antagonistic properties that allow for intoxication of closely related neighboring bacteria via a secreted protein, BcpA. Multiple unique CDI systems can be found in the same bacterial strain, and here we show that these distinct systems interact in several Bcc species. Our findings suggest that the interaction between CDI system proteins is important for interbacterial toxicity. Understanding the mechanism of interplay between CDI systems provides further insight into the complexity of bacterial antagonism. Moreover, since many bacterial species are predicted to encode multiple CDI systems, this study suggests that interactions between these distinct systems likely contribute to the overall competitive fitness of these species.

Functional characterization and clinical implication of a novel epidemic carbapenem-resistant Acinetobacter baumannii genetic marker

ObjectivesThe aim of the study was to evaluate the distribution and function of contact-dependent growth inhibition (CDI) systems associated with carbapenem-resistant Acinetobacter baumannii (CRAB) isolates. MethodsIsolates were examined by multilocus sequence typing (MLST) and polymerase chain reaction (PCR) for the presence of CDI genes in CRAB and carbapenem-susceptible A. baumannii (CSAB) from patients with invasive disease in a medical center in Taiwan. Inter-bacterial competition assays were performed to characterize the in vitro function of the CDI system. ResultsA total of 89 (61.0%) CSAB and 57 (39.0%) CRAB isolates were collected and examined. ST787 (20/57; 35.1%) was the predominant sequence type among CRAB, followed by ST455 (10/57; 17.5%). More than half the CRAB (56.1%, 32/57) belonged to CC455 and more than one third (38.6%, 22/57) to CC92. A novel CDI system, cdiTYTH1, was found in 87.7% (50/57) of the CRAB but in only 1.1% (1/89) of the CSAB isolates (P<0.00001). The cdiTYTH1 was also identified in 94.4% (17/18) of previously genome-sequenced CRAB isolates and only one CSAB isolate from Taiwan. Two other previously reported CDI (cdi19606-1 and cdi19606-2) were not found in these isolates, except both were found in one CSAB. All six CRAB without cdiTYTH1 showed growth inhibition by a CSAB carrying cdiTYTH1 in vitro. All clinical CRAB isolates belonging to the predominant CC455 carried the newly identified cdiTYTH1. ConclusionsThis CDI system was widespread in CRAB clinical isolates and appeared to be an epidemic genetic marker for CRAB in Taiwan. The cdiTYTH1 was functional in vitro in bacterial competition assay.

R-PTP-κ Inhibits Contact-Dependent Cell Growth by Suppressing E2F Activity.

Density-dependent regulation of cell growth is presumed to be caused by cell-cell contact, but the underlying molecular mechanism is not yet clearly defined. Here, we report that receptor-type protein tyrosine phosphatase-kappa (R-PTP-κ) is an important regulator of cell contact-dependent growth inhibition. R-PTP-κ expression increased in proportion to cell density. siRNA-mediated R-PTP-κ downregulation led to the loss of cell contact-mediated growth inhibition, whereas its upregulation reduced anchorage-independent cell growth in soft agar as well as tumor growth in nude mice. Expression profiling and luciferase reporter system-mediated signaling pathway analysis revealed that R-PTP-κ induced under cell contact conditions distinctly suppressed E2F activity. Among the structural domains of R-PTP-κ, the cytoplasmic domain containing the tandemly repeated PTP motif acts as a potent downregulator of the E2F pathway. Specifically, R-PTP-κ suppressed CDK2 activity through the induction of p21Cip1/WAF-1 and p27Kip1, resulting in cell cycle arrest at the G1 phase. In transcriptome-based public datasets generated from four different tumor types, R-PTP-κ expression was negatively correlated with the expression pattern and prognostic value of two known E2F1 target genes (CCNE1 and CDC25A). Therefore, our results indicate that the R-PTP-κ-E2F axis plays a crucial role in cell growth-inhibitory signaling arising from cell-cell contact conditions.

Recipient Cell Factors Influence Interbacterial Competition Mediated by Two Distinct Burkholderia dolosa Contact-Dependent Growth Inhibition Systems.

ABSTRACTContact-dependent growth inhibition (CDI) systems mediate interbacterial antagonism between Gram-negative bacteria by delivering the toxic portion of a large surface protein (termed BcpA in Burkholderia species) to the cytoplasm of neighboring bacteria. Translocation of the antibacterial polypeptide into recipient cells requires specific recipient outer and inner membrane proteins, but the identity of these factors outside several model organisms is unknown. To identify genes involved in CDI susceptibility in the Burkholderia cepacia complex member Burkholderia dolosa, a transposon mutagenesis selection approach was used to enrich for mutants resistant to BcpA-1 or BcpA-2. Subsequent analysis showed that candidate regulatory genes contributed modestly to recipient cell susceptibility to B. dolosa CDI. However, most candidate deletion mutants did not show the same phenotypes as the corresponding transposon mutants. Whole-genome resequencing revealed that these transposon mutants also contained unique mutations within a three gene locus (wabO, BDAG_01006, and BDAG_01005) encoding predicted lipopolysaccharide (LPS) biosynthesis enzymes. B. dolosa wabO, BDAG_01006, or BDAG_01005 mutants were resistant to CDI and produced LPS with altered core oligosaccharide and O-antigen. Although BcpA-1 and BcpA-2 are dissimilar and expected to utilize different outer membrane receptors, intoxication by both proteins was similarly impacted by LPS changes. Together, these findings suggest that alterations in cellular regulation may indirectly impact the efficiency of CDI-mediated competition and demonstrate that LPS is required for intoxication by two distinct B. dolosa BcpA proteins.IMPORTANCEContact-dependent growth inhibition (CDI) system proteins, produced by many Gram-negative bacteria, are narrow spectrum antimicrobials that inhibit the growth of closely related neighboring bacteria. Here, we use the opportunistic pathogen Burkholderia dolosa to identify genes required for intoxication by two distinct CDI system proteins. Our findings suggest that B. dolosa recipient cells targeted by CDI systems are only intoxicated if they produce full-length lipopolysaccharide. Understanding the mechanisms underlying antagonistic interbacterial interactions may contribute to future therapeutic development.

Cryptic Genes for Interbacterial Antagonism Distinguish Rickettsia Species Infecting Blacklegged Ticks From Other Rickettsia Pathogens.

BackgroundThe genus Rickettsia (Alphaproteobacteria: Rickettsiales) encompasses numerous obligate intracellular species with predominantly ciliate and arthropod hosts. Notable species are pathogens transmitted to mammals by blood-feeding arthropods. Mammalian pathogenicity evolved from basal, non-pathogenic host-associations; however, some non-pathogens are closely related to pathogens. One such species, Rickettsia buchneri, is prevalent in the blacklegged tick, Ixodes scapularis. While I. scapularis transmits several pathogens to humans, it does not transmit Rickettsia pathogens. We hypothesize that R. buchneri established a mutualism with I. scapularis, blocking tick superinfection with Rickettsia pathogens.MethodsTo improve estimates for assessing R. buchneri infection frequency in blacklegged tick populations, we used comparative genomics to identify an R. buchneri gene (REIS_1424) not present in other Rickettsia species present throughout the I. scapularis geographic range. Bioinformatic and phylogenomics approaches were employed to propose a function for the hypothetical protein (263 aa) encoded by REIS_1424.ResultsREIS_1424 has few analogs in other Rickettsiales genomes and greatest similarity to non-Proteobacteria proteins. This cohort of proteins varies greatly in size and domain composition, possessing characteristics of Recombination hotspot (Rhs) and contact dependent growth inhibition (CDI) toxins, with similarity limited to proximal C-termini (~145 aa). This domain was named CDI-like/Rhs-like C-terminal toxin (CRCT). As such proteins are often found as toxin-antidote (TA) modules, we interrogated REIS_1423 (151 aa) as a putative antidote. Indeed, REIS_1423 is similar to proteins encoded upstream of CRCT domain-containing proteins. Accordingly, we named these proteins CDI-like/Rhs-like C-terminal toxin antidotes (CRCA). R. buchneri expressed both REIS_1423 and REIS_1424 in tick cell culture, and PCR assays showed specificity for R. buchneri over other rickettsiae and utility for positive detection in three tick populations. Finally, phylogenomics analyses uncovered divergent CRCT/CRCA modules in varying states of conservation; however, only R. buchneri and related Tamurae/Ixodes Group rickettsiae carry complete TA modules.ConclusionWe hypothesize that Rickettsia CRCT/CRCA modules circulate in the Rickettsia mobile gene pool, arming rickettsiae for battle over arthropod colonization. While its functional significance remains to be tested, R. buchneri CRCT/CRCA serves as a marker to positively identify infection and begin deciphering the role this endosymbiont plays in the biology of the blacklegged tick.

Functional and Structural Diversity of Bacterial Contact-Dependent Growth Inhibition Effectors.

Bacteria live in complex communities and environments, competing for space and nutrients. Within their niche habitats, bacteria have developed various inter-bacterial mechanisms to compete and communicate. One such mechanism is contact-dependent growth inhibition (CDI). CDI is found in many Gram-negative bacteria, including several pathogens. These CDI+ bacteria encode a CdiB/CdiA two-partner secretion system that delivers inhibitory toxins into neighboring cells upon contact. Toxin translocation results in the growth inhibition of closely related strains and provides a competitive advantage to the CDI+ bacteria. CdiB, an outer-membrane protein, secretes CdiA onto the surface of the CDI+ bacteria. When CdiA interacts with specific target-cell receptors, CdiA delivers its C-terminal toxin region (CdiA-CT) into the target-cell. CdiA-CT toxin proteins display a diverse range of toxic functions, such as DNase, RNase, or pore-forming toxin activity. CDI+ bacteria also encode an immunity protein, CdiI, that specifically binds and neutralizes its cognate CdiA-CT, protecting the CDI+ bacteria from auto-inhibition. In Gram-negative bacteria, toxin/immunity (CdiA-CT/CdiI) pairs have highly variable sequences and functions, with over 130 predicted divergent toxin/immunity complex families. In this review, we will discuss biochemical and structural advances made in the characterization of CDI. This review will focus on the diverse array of CDI toxin/immunity complex structures together with their distinct toxin functions. Additionally, we will discuss the most recent studies on target-cell recognition and toxin entry, along with the discovery of a new member of the CDI loci. Finally, we will offer insights into how these diverse toxin/immunity complexes could be harnessed to fight human diseases.

Mechanistic insights into tRNA cleavage by a contact-dependent growth inhibitor protein and translation factors.

Contact-dependent growth inhibition is a mechanism of interbacterial competition mediated by delivery of the C-terminal toxin domain of CdiA protein (CdiA–CT) into neighboring bacteria. The CdiA–CT of enterohemorrhagic Escherichia coli EC869 (CdiA–CTEC869) cleaves the 3′-acceptor regions of specific tRNAs in a reaction that requires the translation factors Tu/Ts and GTP. Here, we show that CdiA–CTEC869 has an intrinsic ability to recognize a specific sequence in substrate tRNAs, and Tu:Ts complex promotes tRNA cleavage by CdiA–CTEC869. Uncharged and aminoacylated tRNAs (aa-tRNAs) were cleaved by CdiA–CTEC869 to the same extent in the presence of Tu/Ts, and the CdiA–CTEC869:Tu:Ts:tRNA(aa-tRNA) complex formed in the presence of GTP. CdiA–CTEC869 interacts with domain II of Tu, thereby preventing the 3′-moiety of tRNA to bind to Tu as in canonical Tu:GTP:aa-tRNA complexes. Superimposition of the Tu:GTP:aa-tRNA structure onto the CdiA–CTEC869:Tu structure suggests that the 3′-portion of tRNA relocates into the CdiA–CTEC869 active site, located on the opposite side to the CdiA–CTEC869 :Tu interface, for tRNA cleavage. Thus, CdiA–CTEC869 is recruited to Tu:GTP:Ts, and CdiA–CT:Tu:GTP:Ts recognizes substrate tRNAs and cleaves them. Tu:GTP:Ts serves as a reaction scaffold that increases the affinity of CdiA–CTEC869 for substrate tRNAs and induces a structural change of tRNAs for efficient cleavage by CdiA–CTEC869.

Comparative genome analysis, predicted lifestyle and antimicrobial strategies of Lactococcus carnosus and Lactococcus paracarnosus isolated from meat

Strains of the psychrotrophic bacterium Lactococcus piscium have gained increasing attention as potentially bioprotective cultures due to their assertiveness against fish and meat spoilage bacteria as well as pathogenic bacteria. Recently, we have described two novel species within the genus Lactococcus (Lc.) namely Lc. carnosus (TMW 2.1612T) and Lc. paracarnosus (TMW 2.1615T) isolated from modified atmosphere packaged meat. Within this study, we compared the genomes of two Lc. carnosus strains, two Lc. paracarnosus strains and 16 Lc. piscium strains from our laboratory and five publicly available genomes previously affiliated to the species Lc. piscium. Our phylogenetic analysis supports reclassification of 20 of the strains to either Lc. carnosus or Lc. paracarnosus, so far limiting the Lc. piscium type strain (DSM 6634T) as sole representative of this species. Comparative genomics approach was conducted to predict underlying mechanisms involved in interspecies competition strategies of Lc. carnosus and Lc. paracarnosus against meat spoilers and predict their lifestyle in meat environments. In general, strains of the three species were highly similar regarding metabolic pathways for most of the relevant meat-derived substrates. In silico analyses enabled prediction of homolactic hexose fermentation by Lc. carnosus, Lc. paracarnosus and Lc. piscium. Further, genes required for the heterofermentative metabolism of hexoses and pentoses were only found in the Lc. pisicum type strain (DSM 6634T). We predict a low spoilage potential for Lc. carnosus and Lc. paracarnosus strains. No genes for decarboxylation of amino acids yielding biogenic amines were found in the genomes. Regarding their antimicrobial mechanisms against spoilers, we found a strain-specific putative polymorphic toxin system predictively delivered by the type VIIb secretion system, enabling cell-to-cell contact-dependent growth inhibition. Furthermore, we found additional genes predictively involved to the suppression of spoilers within the food microbiome (prophages, lytic domains, bacteriocins, metabolites).

Mounting, structure and autocleavage of a type VI secretion-associated Rhs polymorphic toxin

Bacteria have evolved toxins to outcompete other bacteria or to hijack host cell pathways. One broad family of bacterial polymorphic toxins gathers multidomain proteins with a modular organization, comprising a C-terminal toxin domain fused to a N-terminal domain that adapts to the delivery apparatus. Polymorphic toxins include bacteriocins, contact-dependent growth inhibition systems, and specialized Hcp, VgrG, PAAR or Rhs Type VI secretion (T6SS) components. We recently described and characterized Tre23, a toxin domain fused to a T6SS-associated Rhs protein in Photorhabdus laumondii, Rhs1. Here, we show that Rhs1 forms a complex with the T6SS spike protein VgrG and the EagR chaperone. Using truncation derivatives and cross-linking mass spectrometry, we demonstrate that VgrG-EagR-Rhs1 complex formation requires the VgrG C-terminal β-helix and the Rhs1 N-terminal region. We then report the cryo-electron-microscopy structure of the Rhs1-EagR complex, demonstrating that the Rhs1 central region forms a β-barrel cage-like structure that encapsulates the C-terminal toxin domain, and provide evidence for processing of the Rhs1 protein through aspartyl autoproteolysis. We propose a model for Rhs1 loading on the T6SS, transport and delivery into the target cell.

Lipidation of Class IV CdiA Effector Proteins Promotes Target Cell Recognition during Contact-Dependent Growth Inhibition.

ABSTRACTContact-dependent growth inhibition (CDI) systems enable the direct transfer of protein toxins between competing Gram-negative bacteria. CDI+ strains produce cell surface CdiA effector proteins that bind specific receptors on neighboring bacteria to initiate toxin delivery. Three classes of CdiA effectors that recognize different outer membrane protein receptors have been characterized in Escherichia coli to date. Here, we describe a fourth effector class that uses the lipopolysaccharide (LPS) core as a receptor to identify target bacteria. Selection for CDI-resistant target cells yielded waaF and waaP “deep-rough” mutants, which are unable to synthesize the full LPS core. The CDI resistance phenotypes of other waa mutants suggest that phosphorylated inner-core heptose residues form a critical CdiA recognition epitope. Class IV cdi loci also encode putative lysyl acyltransferases (CdiC) that are homologous to enzymes that lipidate repeats-in-toxin (RTX) cytolysins. We found that catalytically active CdiC is required for full target cell killing activity, and we provide evidence that the acyltransferase appends 3-hydroxydecanoate to a specific Lys residue within the CdiA receptor-binding domain. We propose that the lipid moiety inserts into the hydrophobic leaflet of lipid A to anchor CdiA interactions with the core oligosaccharide. Thus, LPS-binding CDI systems appear to have co-opted an RTX toxin-activating acyltransferase to increase the affinity of CdiA effectors for the target cell outer membrane.

Burkholderia multivorans requires species-specific GltJK for entry of a contact-dependent growth inhibition system protein.

Interbacterial antagonism and communication are driving forces behind microbial community development. In many Gram‐negative bacteria, contact‐dependent growth inhibition (CDI) systems contribute to these microbial interactions. CDI systems deliver the toxic C‐terminus of a large surface exposed protein to the cytoplasm of neighboring bacteria upon cell−contact. Termed the BcpA‐CT, import of this toxic effector domain is mediated by specific, yet largely unknown receptors on the recipient cell outer and inner membranes. In this study, we demonstrated that cytoplasmic membrane proteins GltJK, components of a predicted ABC‐type transporter, are required for entry of CDI system protein BcpA‐2 into Burkholderia multivorans recipient cells. Consistent with current CDI models, gltJK were also required for recipient cell susceptibility to a distinct BcpA‐CT that shared sequences within the predicted “translocation domain” of BcpA‐2. Strikingly, this translocation domain showed low sequence identity to the analogous region of an Escherichia coli GltJK‐utilizing CDI system protein. Our results demonstrated that recipient bacteria expressing E. coli gltJK were resistant to BcpA‐2‐mediated interbacterial antagonism, suggesting that BcpA‐2 specifically recognizes Burkholderia GltJK. Using a series of chimeric proteins, the specificity determinant was mapped to Burkholderia‐specific sequences at the GltK C‐terminus, providing insight into BcpA transport across the recipient cell cytoplasmic membrane.

Escherichia coli EC93 deploys two plasmid-encoded class I contact-dependent growth inhibition systems for antagonistic bacterial interactions.

The phenomenon of contact-dependent growth inhibition (CDI) and the genes required for CDI (cdiBAI) were identified and isolated in 2005 from an Escherichia coli isolate (EC93) from rats. Although the cdiBAI EC93 locus has been the focus of extensive research during the past 15 years, little is known about the EC93 isolate from which it originates. Here we sequenced the EC93 genome and find two complete and functional cdiBAI loci (including the previously identified cdi locus), both carried on a large 127 kb plasmid. These cdiBAI systems are differentially expressed in laboratory media, enabling EC93 to outcompete E. coli cells lacking cognate cdiI immunity genes. The two CDI systems deliver distinct effector peptides that each dissipate the membrane potential of target cells, although the two toxins display different toxic potencies. Despite the differential expression and toxic potencies of these CDI systems, both yielded similar competitive advantages against E. coli cells lacking immunity. This can be explained by the fact that the less expressed cdiBAI system (cdiBAIEC93-2) delivers a more potent toxin than the highly expressed cdiBAIEC93-1 system. Moreover, our results indicate that unlike most sequenced CDI+ bacterial isolates, the two cdi loci of E. coli EC93 are located on a plasmid and are expressed in laboratory media.

Contact-Dependent Growth Inhibition in Bacteria: Do Not Get Too Close!

Over millions of years of evolution, bacteria have developed complex strategies for intra-and interspecies interactions and competition for ecological niches and resources. Contact-dependent growth inhibition systems (CDI) are designed to realize a direct physical contact of one bacterial cell with other cells in proximity via receptor-mediated toxin delivery. These systems are found in many microorganisms including clinically important human pathogens. The main purpose of these systems is to provide competitive advantages for the growth of the population. In addition, non-competitive roles for CDI toxin delivery systems including interbacterial signal transduction and mediators of bacterial collaboration have been suggested. In this review, our goal was to systematize the recent findings on the structure, mechanisms, and purpose of CDI systems in bacterial populations and discuss the potential biological and evolutionary impact of CDI-mediated interbacterial competition and/or cooperation.

Structural insight into toxin secretion by contact-dependent growth inhibition transporters

Bacterial contact-dependent growth inhibition (CDI) systems use a type Vb secretion mechanism to export large CdiA toxins across the outer membrane by dedicated outer membrane transporters called CdiB. Here, we report the first crystal structures of two CdiB transporters from Acinetobacter baumannii and Escherichia coli. CdiB transporters adopt a TpsB fold, containing a 16-stranded transmembrane β-barrel connected to two periplasmic domains. The lumen of the CdiB pore is occluded by an N-terminal α-helix and the conserved extracellular loop 6; these two elements adopt different conformations in the structures. We identified a conserved DxxG motif located on strand β1 that connects loop 6 through different networks of interactions. Structural modifications of DxxG induce rearrangement of extracellular loops and alter interactions with the N-terminal α-helix, preparing the system for α-helix ejection. Using structural biology, functional assays, and molecular dynamics simulations, we show how the barrel pore is primed for CdiA toxin secretion.

Abstract 3572: Merlin deficiency induces redox imbalance in breast cancer

Abstract Merlin is encoded by the NF2 gene and, as a tumor suppressor, stalls cell proliferation by contact-dependent growth inhibition. Prior work from our laboratory has established that Merlin levels are significantly decreased in breast tumor tissues representing advanced grade. Moreover, Merlin expression is completely lost in cases representing metastatic breast cancer. Therefore, we mimicked Merlin deficiency by stably knocking down NF2 in non-metastatic breast cancer cells. Recognizing the role of metabolic reprogramming in tumor progression, we undertook untargeted metabolomics analysis - this effort identified low levels of reduced glutathione (GSH) in Merlin-deficient cells. Given the role of GSH as the most abundant antioxidant cofactor, we explored if a major reactive oxygen species (ROS) clearance mechanism is compromised in Merlin-deficient breast cancer cells. Indeed, accumulated levels of ROS were detected in Merlin-deficient cells, suggesting that Merlin deficiency causes redox imbalance. Quenching of elevated ROS levels attenuated migration and invasion capabilities indicating a role of ROS in intensifying malignant behaviors upon Merlin deficiency. Furthermore, dysregulated STAT3 activity upregulated the expression of NADPH oxidase (NOX) enzymes in Merlin-deficient breast cancer cells compared to their control counterparts. These findings were also recapitulated in non-tumorigenic Nf2 −/- mouse embryonic fibroblasts further supporting the key role of Merlin in these phenotypes. In conclusion, ROS accumulation and increased malignancy in Merlin-deficient cells may be the result of a dysregulated cellular redox management system. Citation Format: Mateus Mota, Brandon Metge, Lalita Shevde-Samant. Merlin deficiency induces redox imbalance in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3572.

Capsule Protects Acinetobacter baumannii From Inter-Bacterial Competition Mediated by CdiA Toxin.

Currently, Acinetobacter baumannii is considered as one of the most important infectious agents causing hospital acquired infections worldwide. It has been observed that many clinically important pathogens express contact-dependent growth inhibition (CDI) phenomenon, which modulates cell–cell and cell–environment interactions, potentially allowing bacteria to adapt to ever-changing conditions. Mainly, these systems are used for the inhibition of the growth of genetically different individuals within the same species. In this work, by performing cell competition assays with three genotypically different (as determined by pulse-field gel electrophoresis) clinical A. baumannii isolates II-c, II-a, and II-a1, we show that A. baumannii capsule is the main feature protecting from CDI-mediated inhibition. We also observed that for one clinical isolate, the two-component BfmRS system, contributed to the resistance against CDI-mediated inhibition. Moreover, we were able to demonstrate, that the effector protein CdiA is released into the growth media and exhibits its inhibitory activity without the requirement of a cell–cell contact. Lastly, by evaluating the remaining number of the cells pre-mixed with the CdiA and performing live/dead assay, we demonstrate that purified CdiA protein causes a rapid cell growth arrest. Our results indicate, that capsule efficiently protects A. baumannii from a CDI-mediated inhibition by a clinical A. baumannii V15 strain, which is able to secrete CdiA effector into the growth media and cause target cell growth arrest without a cell–cell contact.

Identification of A Putative T6SS Immunity Islet in Salmonella Typhi.

Typhoid fever is a major global health problem and is the result of systemic infections caused by the human-adapted bacterial pathogen Salmonella enterica serovar Typhi (S. Typhi). The pathology underlying S. Typhi infections significantly differ from infections caused by broad host range serovars of the same species, which are a common cause of gastroenteritis. Accordingly, identifying S. Typhi genetic factors that impart functionality absent from broad host range serovars offers insights into its unique biology. Here, we used an in-silico approach to explore the function of an uncharacterized 14-gene S. Typhi genomic islet. Our results indicated that this islet was specific to the S. enterica species, where it was encoded by the Typhi and Paratyphi A serovars, but was generally absent from non-typhoidal serovars. Evidence was gathered using comparative genomics and sequence analysis tools, and indicated that this islet was comprised of Type VI secretion system (T6SS) and contact-dependent growth inhibition (CDI) genes, the majority of which appeared to encode orphan immunity proteins that protected against the activities of effectors and toxins absent from the S. Typhi genome. We herein propose that this islet represents an immune system that protects S. Typhi against competing bacteria within the human gut.

A comparative genomics approach identifies contact-dependent growth inhibition as a virulence determinant

Emerging evidence suggests the Pseudomonas aeruginosa accessory genome is enriched with uncharacterized virulence genes. Identification and characterization of such genes may reveal novel pathogenic mechanisms used by particularly virulent isolates. Here, we utilized a mouse bacteremia model to quantify the virulence of 100 individual P. aeruginosa bloodstream isolates and performed whole-genome sequencing to identify accessory genomic elements correlated with increased bacterial virulence. From this work, we identified a specific contact-dependent growth inhibition (CDI) system enriched among highly virulent P. aeruginosa isolates. CDI systems contain a large exoprotein (CdiA) with a C-terminal toxin (CT) domain that can vary between different isolates within a species. Prior work has revealed that delivery of a CdiA-CT domain upon direct cell-to-cell contact can inhibit replication of a susceptible target bacterium. Aside from mediating interbacterial competition, we observed our virulence-associated CdiA-CT domain to promote toxicity against mammalian cells in culture and lethality during mouse bacteremia. Structural and functional studies revealed this CdiA-CT domain to have in vitro tRNase activity, and mutations that abrogated this tRNAse activity in vitro also attenuated virulence. Furthermore, CdiA contributed to virulence in mice even in the absence of contact-dependent signaling. Overall, our findings indicate that this P. aeruginosa CDI system functions as both an interbacterial inhibition system and a bacterial virulence factor against a mammalian host. These findings provide an impetus for continued studies into the complex role of CDI systems in P. aeruginosa pathogenesis.