Glucose confers protection to Escherichia coli against contact killing by Vibrio cholerae.

Evolutionary arms races are broadly prevalent among organisms including bacteria, which have evolved defensive strategies against various attackers. A common microbial aggression mechanism is the type VI secretion system (T6SS), a contact-dependent bacterial weapon used to deliver toxic effector proteins into adjacent target cells. Sibling cells constitutively express immunity proteins that neutralize effectors. However, less is known about factors that protect non-sibling bacteria from T6SS attacks independently of cognate immunity proteins. In this study, we observe that human Escherichia coli commensal strains sensitive to T6SS attacks from Vibrio cholerae are protected when co-cultured with glucose. We confirm that glucose does not impair V. cholerae T6SS activity. Instead, we find that cells lacking the cAMP receptor protein (CRP), which regulates expression of hundreds of genes in response to glucose, survive significantly better against V. cholerae T6SS attacks even in the absence of glucose. Finally, we show that the glucose-mediated T6SS protection varies with different targets and killers. Our findings highlight the first example of an extracellular small molecule modulating a genetically controlled response for protection against T6SS attacks. This discovery may have major implications for microbial interactions during pathogen-host colonization and survival of bacteria in environmental communities.

A family of Type VI secretion system effector proteins that form ion-selective pores.

Type VI secretion systems (T6SSs) are nanomachines widely used by bacteria to deliver toxic effector proteins directly into neighbouring cells. However, the modes of action of many effectors remain unknown. Here we report that Ssp6, an anti-bacterial effector delivered by a T6SS of the opportunistic pathogen Serratia marcescens, is a toxin that forms ion-selective pores. Ssp6 inhibits bacterial growth by causing depolarisation of the inner membrane in intoxicated cells, together with increased outer membrane permeability. Reconstruction of Ssp6 activity in vitro demonstrates that it forms cation-selective pores. A survey of bacterial genomes reveals that genes encoding Ssp6-like effectors are widespread in Enterobacteriaceae and often linked with T6SS genes. We conclude that Ssp6 and similar proteins represent a new family of T6SS-delivered anti-bacterial effectors.

The Antibacterial and Anti-Eukaryotic Type VI Secretion System MIX-Effector Repertoire in Vibrionaceae.

Vibrionaceae is a widespread family of aquatic bacteria that includes emerging pathogens and symbionts. Many Vibrionaceae harbor a type VI secretion system (T6SS), which is a secretion apparatus used to deliver toxins, termed effectors, into neighboring cells. T6SSs mediate both antibacterial and anti-eukaryotic activities. Notably, antibacterial effectors are encoded together with a gene that encodes a cognate immunity protein so as to antagonize the toxicity of the effector. The MIX (Marker for type sIX effectors) domain has been previously defined as a marker of T6SS effectors carrying polymorphic C-terminal toxins. Here, we set out to identify the Vibrionaceae MIX-effector repertoire and to analyze the various toxin domains they carry. We used a computational approach to search for the MIX-effectors in the Vibrionaceae genomes, and grouped them into clusters based on the C-terminal toxin domains. We classified MIX-effectors as either antibacterial or anti-eukaryotic, based on the presence or absence of adjacent putative immunity genes, respectively. Antibacterial MIX-effectors carrying pore-forming, phospholipase, nuclease, peptidoglycan hydrolase, and protease activities were found. Furthermore, we uncovered novel virulence MIX-effectors. These are encoded by "professional MIXologist" strains that employ a cocktail of antibacterial and anti-eukaryotic MIX-effectors. Our findings suggest that certain Vibrionaceae adapted their antibacterial T6SS to mediate interactions with eukaryotic hosts or predators.

Diversification of Type VI Secretion System Toxins Reveals Ancient Antagonism among Bee Gut Microbes.

Microbial communities are shaped by interactions among their constituent members. Some Gram-negative bacteria employ type VI secretion systems (T6SSs) to inject protein toxins into neighboring cells. These interactions have been theorized to affect the composition of host-associated microbiomes, but the role of T6SSs in the evolution of gut communities is not well understood. We report the discovery of two T6SSs and numerous T6SS-associated Rhs toxins within the gut bacteria of honey bees and bumble bees. We sequenced the genomes of 28 strains of Snodgrassella alvi, a characteristic bee gut microbe, and found tremendous variability in their Rhs toxin complements: altogether, these strains appear to encode hundreds of unique toxins. Some toxins are shared with Gilliamella apicola, a coresident gut symbiont, implicating horizontal gene transfer as a source of toxin diversity in the bee gut. We use data from a transposon mutagenesis screen to identify toxins with antibacterial function in the bee gut and validate the function and specificity of a subset of these toxin and immunity genes in Escherichia coli Using transcriptome sequencing, we demonstrate that S. alvi T6SSs and associated toxins are upregulated in the gut environment. We find that S. alvi Rhs loci have a conserved architecture, consistent with the C-terminal displacement model of toxin diversification, with Rhs toxins, toxin fragments, and cognate immunity genes that are expressed and confer strong fitness effects in vivo Our findings of T6SS activity and Rhs toxin diversity suggest that T6SS-mediated competition may be an important driver of coevolution within the bee gut microbiota.IMPORTANCE The structure and composition of host-associated bacterial communities are of broad interest, because these communities affect host health. Bees have a simple, conserved gut microbiota, which provides an opportunity to explore interactions between species that have coevolved within their host over millions of years. This study examined the role of type VI secretion systems (T6SSs)-protein complexes used to deliver toxic proteins into bacterial competitors-within the bee gut microbiota. We identified two T6SSs and diverse T6SS-associated toxins in bacterial strains from bees. Expression of these genes is increased in bacteria in the bee gut, and toxin and immunity genes demonstrate antibacterial and protective functions, respectively, when expressed in Escherichia coli Our results suggest that coevolution among bacterial species in the bee gut has favored toxin diversification and maintenance of T6SS machinery, and demonstrate the importance of antagonistic interactions within host-associated microbial communities.