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.

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

Contact-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.

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.

Burkholderia cepacia Complex Contact-Dependent Growth Inhibition Systems Mediate Interbacterial Competition.

Burkholderia species, including opportunistic pathogens in the Burkholderia cepacia complex (Bcc), have genes to produce contact-dependent growth inhibition (CDI) system proteins. CDI is a phenomenon in which Gram-negative bacteria use the toxic C terminus of a polymorphic surface-exposed exoprotein, BcpA, to inhibit the growth of susceptible bacteria upon direct cell-cell contact. Production of a small immunity protein, BcpI, prevents autoinhibition. Although CDI systems appear widespread in Gram-negative bacteria, their function has been primarily examined in several model species. Here we demonstrate that genes encoding predicted CDI systems in Bcc species exhibit considerable diversity. We also show that Burkholderia multivorans, which causes pulmonary infections in patients with cystic fibrosis, expresses genes that encode two CDI systems, both of which appear distinct from the typical Burkholderia-type CDI system. Each system can mediate intrastrain interbacterial competition and contributes to bacterial adherence. Surprisingly, the immunity-protein-encoding bcpI gene of CDI system 1 could be mutated without obvious deleterious effects. We also show that nonpathogenic Burkholderia thailandensis uses CDI to control B. multivorans growth during coculture, providing one of the first examples of interspecies CDI and suggesting that CDI systems could be manipulated to develop therapeutic strategies targeting Bcc pathogens.IMPORTANCE Competition among bacteria affects microbial colonization of environmental niches and host organisms, particularly during polymicrobial infections. The Bcc is a group of environmental bacteria that can cause life-threatening opportunistic infections in patients who have cystic fibrosis or are immunocompromised. Understanding the mechanisms used by these bacterial pathogens to compete with one another may lead to the development of more effective therapies. Findings presented here demonstrate that a Bcc species, Burkholderia multivorans, produces functional CDI system proteins and that growth of this pathogen can be controlled by CDI system proteins produced by neighboring Burkholderia cells.

Contact-dependent interbacterial toxins deliver a message.

Both Gram-negative and Gram-positive organisms harbor systems for delivering toxins to neighboring bacteria upon direct cell contact. These systems, typified by type VI secretion (T6S) and contact-dependent growth inhibition (CDI) systems, are defined by their ability to mediate interbacterial competition in vitro, while their biological roles have remained uncertain. Recent research into the mechanisms of toxin delivery and activity, as well as investigation of contact-dependent toxin function during relevant biological processes, has offered insight into how interbacterial competition might work outside of the laboratory. Furthermore, non-competitive roles for contact-dependent toxin delivery systems, including interbacterial signal transduction, have been described. This review suggests that contact-dependent toxin delivery systems that exhibit functions beyond interbacterial competition are probably more common than currently appreciated.

Burkholderia thailandensis: Genetic Manipulation.

Burkholderia thailandensis is a Gram-negative bacterium endemic to Southeast Asian and northern Australian soils. It is non-pathogenic; therefore, it is commonly used as a model organism for the related human pathogens Burkholderia mallei and Burkholderia pseudomallei. B. thailandensis is relatively easily genetically manipulated and a variety of robust genetic tools can be used in this organism. This unit describes protocols for conjugation, natural transformation, mini-Tn7 insertion, and allelic exchange in B. thailandensis. © 2017 by John Wiley & Sons, Inc.

Are CDI Systems Multicolored, Facultative, Helping Greenbeards?

Competitive and cooperative interactions between organisms, including bacteria, can significantly impact the composition of a community and the fitness of its members, as well as the fitness of their hosts when communities are living on or within other organisms. Understanding the underlying mechanisms is critical to the development of strategies to control microbiological communities that impact animal and plant health and also for understanding the evolution of social behaviors, which has been challenging for evolutionary biologists. Contact-dependent growth inhibition (CDI) is a phenomenon defined by the delivery of a protein toxin to the cytoplasm of neighboring bacteria upon cell-cell contact, resulting in growth inhibition or death unless a specific immunity protein is present. CDI was first described based on observations of interbacterial killing and has been assumed to function primarily as a means of eliminating competitor cells. However, recent molecular evidence indicates that multiple levels of specificity restrict CDI toxin delivery and activity to the same bacterial strain, and that CDI system proteins can mediate cooperative behaviors among 'self' cells, a phenomenon called contact-dependent signaling (CDS). Here we review these recent findings and discuss potential biological and evolutionary implications of CDI system-mediated interbacterial competition and cooperation.

Burkholderia thailandensis: Growth and Laboratory Maintenance.

Burkholderia thailandensis is a nonpathogenic Gram-negative bacterium found in tropical soils. Closely related to several human pathogens, its ease of genetic manipulation, rapid growth in the laboratory, and low virulence make B. thailandensis a commonly used model organism. This unit describes the fundamental protocols for in vitro growth and maintenance of B. thailandensis in the laboratory. © 2016 by John Wiley & Sons, Inc.

Interbacterial signaling via Burkholderia contact-dependent growth inhibition system proteins.

In prokaryotes and eukaryotes, cell-cell communication and recognition of self are critical to coordinate multicellular functions. Although kin and kind discrimination are increasingly appreciated to shape naturally occurring microbe populations, the underlying mechanisms that govern these interbacterial interactions are insufficiently understood. Here, we identify a mechanism of interbacterial signal transduction that is mediated by contact-dependent growth inhibition (CDI) system proteins. CDI systems have been characterized by their ability to deliver a polymorphic protein toxin into the cytoplasm of a neighboring bacterium, resulting in growth inhibition or death unless the recipient bacterium produces a corresponding immunity protein. Using the model organism Burkholderia thailandensis, we show that delivery of a catalytically active CDI system toxin to immune (self) bacteria results in gene expression and phenotypic changes within the recipient cells. Termed contact-dependent signaling (CDS), this response promotes biofilm formation and other community-associated behaviors. Engineered strains that are isogenic with B. thailandensis, except the DNA region encoding the toxin and immunity proteins, did not display CDS, whereas a strain of Burkholderia dolosa producing a nearly identical toxin-immunity pair induced signaling in B. thailandensis Our data indicate that bcpAIOB loci confer dual benefits; they direct antagonism toward non-self bacteria and promote cooperation between self bacteria, with self being defined by the bcpAIOB allele and not by genealogic relatedness.

Kind discrimination and competitive exclusion mediated by contact-dependent growth inhibition systems shape biofilm community structure.

Contact-Dependent Growth Inhibition (CDI) is a phenomenon in which bacteria use the toxic C-terminus of a large exoprotein (called BcpA in Burkholderia species) to inhibit the growth of neighboring bacteria upon cell-cell contact. CDI systems are present in a wide range of Gram-negative proteobacteria and a hallmark feature is polymorphism amongst the exoprotein C-termini (BcpA-CT in Burkholderia) and amongst the small immunity proteins (BcpI) that protect against CDI in an allele-specific manner. In addition to CDI, the BcpAIOB proteins of Burkholderia thailandensis mediate biofilm formation, and they do so independent of BcpA-mediated interbacterial competition, suggesting a cooperative role for CDI system proteins in this process. CDI has previously only been demonstrated between CDI+ and CDI- bacteria, leaving the roles of CDI system-mediated interbacterial competition and of CDI system diversity in nature unknown. We constructed B. thailandensis strains that differed only in the BcpA-CT and BcpI proteins they produced. When co-cultured on agar, these strains each participated in CDI and the outcome of the competition depended on both CDI system efficiency and relative bacterial numbers initially. Strains also participated in CDI during biofilm development, resulting in pillar structures that were composed of only a single BcpA-CT/BcpI type. Moreover, a strain producing BcpA-CT/BcpI proteins of one type was prevented from joining a pre-established biofilm community composed of bacteria producing BcpA-CT/BcpI proteins of a different type, unless it also produced the BcpI protein of the established strain. Bacteria can therefore use CDI systems for kind recognition and competitive exclusion of 'non-self' bacteria from a pre-established biofilm. Our data indicate that CDI systems function in both cooperative and competitive behaviors to build microbial communities that are composed of only bacteria that are related via their CDI system alleles.

Burkholderia BcpA mediates biofilm formation independently of interbacterial contact-dependent growth inhibition.

Contact-dependent growth inhibition (CDI) is a phenomenon in which Gram-negative bacteria use the toxic C-terminus of a large surface-exposed exoprotein to inhibit the growth of susceptible bacteria upon cell-cell contact. Little is known about when and where bacteria express the genes encoding CDI system proteins and how these systems contribute to the survival of bacteria in their natural niche. Here we establish that, in addition to mediating interbacterial competition, the Burkholderia thailandensis CDI system exoprotein BcpA is required for biofilm development. We also provide evidence that the catalytic activity of BcpA and extracellular DNA are required for the characteristic biofilm pillars to form. We show using a bcpA-gfp fusion that within the biofilm, expression of the CDI system-encoding genes is below the limit of detection for the majority of bacteria and only a subset of cells express the genes strongly at any given time. Analysis of a strain constitutively expressing the genes indicates that native expression is critical for biofilm architecture. Although CDI systems have so far only been demonstrated to be involved in interbacterial competition, constitutive production of the system's immunity protein in the entire bacterial population did not alter biofilm formation, indicating a CDI-independent role for BcpA in this process. We propose, therefore, that bacteria may use CDI proteins in cooperative behaviours, like building biofilm communities, and in competitive behaviours that prevent non-self bacteria from entering the community.

The Burkholderia bcpAIOB genes define unique classes of two-partner secretion and contact dependent growth inhibition systems.

Microbes have evolved many strategies to adapt to changes in environmental conditions and population structures, including cooperation and competition. One apparently competitive mechanism is contact dependent growth inhibition (CDI). Identified in Escherichia coli, CDI is mediated by Two-Partner Secretion (TPS) pathway proteins, CdiA and CdiB. Upon cell contact, the toxic C-terminus of the TpsA family member CdiA, called the CdiA-CT, inhibits the growth of CDI(-) bacteria. CDI(+) bacteria are protected from autoinhibition by an immunity protein, CdiI. Bioinformatic analyses indicate that CDI systems are widespread amongst α, ß, and γ proteobacteria and that the CdiA-CTs and CdiI proteins are highly variable. CdiI proteins protect against CDI in an allele-specific manner. Here we identify predicted CDI system-encoding loci in species of Burkholderia, Ralstonia and Cupriavidus, named bcpAIOB, that are distinguished from previously-described CDI systems by gene order and the presence of a small ORF, bcpO, located 5' to the gene encoding the TpsB family member. A requirement for bcpO in function of BcpA (the TpsA family member) was demonstrated, indicating that bcpAIOB define a novel class of TPS system. Using fluorescence microscopy and flow cytometry, we show that these genes are expressed in a probabilistic manner during culture of Burkholderia thailandensis in liquid medium. The bcpAIOB genes and extracellular DNA were required for autoaggregation and adherence to an abiotic surface, suggesting that CDI is required for biofilm formation, an activity not previously attributed to CDI. By contrast to what has been observed in E. coli, the B. thailandensis bcpAIOB genes only mediated interbacterial competition on a solid surface. Competition occurred in a defined spatiotemporal manner and was abrogated by allele-specific immunity. Our data indicate that the bcpAIOB genes encode distinct classes of CDI and TPS systems that appear to function in sociomicrobiological community development.

Redundancy and specificity of Escherichia coli iron acquisition systems during urinary tract infection.

Uropathogenic Escherichia coli (UPEC), the predominant cause of uncomplicated urinary tract infection (UTI), utilizes an array of outer membrane iron receptors to facilitate siderophore and heme import from within the iron-limited urinary tract. While these systems are required for UPEC in vivo fitness and are assumed to be functionally redundant, the relative contributions of specific receptors to pathogenesis are unknown. To delineate the relative roles of distinct UPEC iron acquisition systems in UTI, isogenic mutants in UPEC strain CFT073 or 536 lacking individual receptors were competed against one another in vivo in a series of mixed infections. When combinations of up to four mutants were coinoculated using a CBA/J mouse model of ascending UTI, catecholate receptor mutants (ΔfepA, Δiha, and ΔiroN mutants) were equally fit, suggesting redundant function. However, noncatecholate siderophore receptor mutants, including the ΔiutA aerobactin receptor mutant and the ΔfyuA yersiniabactin receptor mutant, were frequently outcompeted by coinoculated mutants, indicating that these systems contribute more significantly to UPEC iron acquisition in vivo. A tissue-specific preference for heme acquisition was also observed, as a heme uptake-deficient Δhma ΔchuA double mutant was outcompeted by siderophore receptor mutants specifically during kidney colonization. The relative contribution of each receptor to UTI only partially correlated with in vivo levels of receptor gene expression, indicating that other factors likely contributed to the observed fitness differences. Overall, our results suggest that UPEC iron receptors provide both functional redundancy and niche specificity for this pathogen as it colonizes distinct sites within the urinary tract.