Ancient Yersinia pestis genomes lack the virulence-associated YpfΦ prophage present in modern pandemic strains.

Yersinia pestis is the causative agent of at least three major plague pandemics (Justinianic, Medieval and Modern). Previous studies on ancient Y. pestis genomes revealed that several genomic alterations had occurred approximately 5000-3000 years ago and contributed to the remarkable virulence of this pathogen. How a subset of strains evolved to cause the Modern pandemic is less well-understood. Here, we examined the virulence-associated prophage (YpfΦ), which had been postulated to be exclusively present in the genomes of strains associated with the Modern pandemic. The analysis of two new Y. pestis genomes from medieval/early modern Denmark confirmed that the phage is absent from the genome of strains dating to this time period. An extended comparative genome analysis of over 300 strains spanning more than 5000 years showed that the prophage is found in the genomes of modern strains only and suggests an integration into the genome during recent Y. pestis evolution. The phage-encoded Zot protein showed structural homology to a virulence factor of Vibrio cholerae. Similar to modern Y. pestis, we observed phages with a common origin to YpfΦ in individual strains of other bacterial species. Our findings present an updated view on the prevalence of YpfΦ, which might contribute to our understanding of the host spectrum, geographical spread and virulence of Y. pestis responsible for the Modern pandemic.

Presence and absence of type VI secretion systems in bacteria.

The type VI secretion system (T6SS) is a molecular puncturing device that enables Gram-negative bacteria to kill competitors, manipulate host cells and take up nutrients. Who would want to miss such superpowers? Indeed, many studies show how widespread the secretion apparatus is among microbes. However, it is becoming evident that, on multiple taxonomic levels, from phyla to species and strains, some bacteria lack a T6SS. Here, we review who does and does not have a type VI secretion apparatus and speculate on the dynamic process of gaining and losing the secretion system to better understand its spread and distribution across the microbial world.

Bacteroides muris sp. nov. isolated from the cecum of wild-derived house mice.

Two bacterial strains, KH365_2T and KH569_7, were isolated from the cecum contents of wild-derived house mice. The strains were characterized as Gram-negative, rod-shaped, strictly anaerobic, and non-motile. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both strains were most closely related to Bacteroides uniformis ATCC 8492T. Whole genome sequences of KH365_2T and KH569_7 strains have a DNA G + C content of 46.02% and 46.03% mol, respectively. Most morphological and biochemical characteristics did not differ between the newly isolated strains and classified Bacteroides strains. However, the average nucleotide identity (ANI) and dDNA-DNA hybridization (dDDH) values clearly distinguished the two strains from described members of the genus Bacteroides. Here, we present the phylogeny, morphology, and physiology of a novel species of the genus Bacteroides and propose the name Bacteroides muris sp. nov., with KH365_2T (DSM 114231T = CCUG 76277T) as type strain.

Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae.

Vibrio cholerae is a diverse species of Gram-negative bacteria, commonly found in the aquatic environment and the causative agent of the potentially deadly disease cholera. These bacteria employ a type VI secretion system (T6SS) when they encounter prokaryotic and eukaryotic competitors. This contractile puncturing device translocates a set of effector proteins into neighboring cells. Translocated effectors are toxic unless the targeted cell produces immunity proteins that bind and deactivate incoming effectors. Comparison of multiple V. cholerae strains indicates that effectors are encoded in T6SS effector modules on mobile genetic elements. We identified a diverse group of chimeric T6SS adaptor proteins required for the translocation of diverse effectors encoded in modules. An example for a T6SS effector that requires T6SS adaptor protein 1 (Tap-1) is TseL found in pandemic V. cholerae O1 serogroup strains and other clinical isolates. We propose a model in which Tap-1 is required for loading TseL onto the secretion apparatus. After T6SS-mediated TseL export is completed, Tap-1 is retained in the bacterial cell to load other T6SS machines.

Dual expression profile of type VI secretion system immunity genes protects pandemic Vibrio cholerae.

The Vibrio cholerae type VI secretion system (T6SS) assembles as a molecular syringe that injects toxic protein effectors into both eukaryotic and prokaryotic cells. We previously reported that the V. cholerae O37 serogroup strain V52 maintains a constitutively active T6SS to kill other Gram-negative bacteria while being immune to attack by kin bacteria. The pandemic O1 El Tor V. cholerae strain C6706 is T6SS-silent under laboratory conditions as it does not produce T6SS structural components and effectors, and fails to kill Escherichia coli prey. Yet, C6706 exhibits full resistance when approached by T6SS-active V52. These findings suggested that an active T6SS is not required for immunity against T6SS-mediated virulence. Here, we describe a dual expression profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 that provides pandemic V. cholerae strains with T6SS immunity and allows T6SS-silent strains to maintain immunity against attacks by T6SS-active bacterial neighbors. The dual expression profile allows transcription of the three genes encoding immunity proteins independently of other T6SS proteins encoded within the same operon. One of these immunity proteins, TsiV2, protects against the T6SS effector VasX which is encoded immediately upstream of tsiV2. VasX is a secreted, lipid-binding protein that we previously characterized with respect to T6SS-mediated virulence towards the social amoeba Dictyostelium discoideum. Our data suggest the presence of an internal promoter in the open reading frame of vasX that drives expression of the downstream gene tsiV2. Furthermore, VasX is shown to act in conjunction with VasW, an accessory protein to VasX, to compromise the inner membrane of prokaryotic target cells. The dual regulatory profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 permits V. cholerae to tightly control T6SS gene expression while maintaining immunity to T6SS activity.

Lytic activity of the Vibrio cholerae type VI secretion toxin VgrG-3 is inhibited by the antitoxin TsaB.

The type VI secretion system (T6SS) of Gram-negative bacteria has been implicated in microbial competition; however, which components serve purely structural roles, and which serve as toxic effectors remains unresolved. Here, we present evidence that VgrG-3 of the Vibrio cholerae T6SS has both structural and toxin activity. Specifically, we demonstrate that the C-terminal extension of VgrG-3 acts to degrade peptidoglycan and hypothesize that this assists in the delivery of accessory T6SS toxins of V. cholerae. To avoid self-intoxication, V. cholerae expresses an anti-toxin encoded immediately downstream of vgrG-3 that inhibits VgrG-3-mediated lysis through direct interaction.

Human tetherin exerts strong selection pressure on the HIV-1 group N Vpu protein.

HIV-1 groups M and N emerged within the last century following two independent cross-species transmissions of SIVcpz from chimpanzees to humans. In contrast to pandemic group M strains, HIV-1 group N viruses are exceedingly rare, with only about a dozen infections identified, all but one in individuals from Cameroon. Poor adaptation to the human host may be responsible for this limited spread of HIV-1 group N in the human population. Here, we analyzed the function of Vpu proteins from seven group N strains from Cameroon, the place where this zoonosis originally emerged. We found that these N-Vpus acquired four amino acid substitutions (E15A, V19A and IV25/26LL) in their transmembrane domain (TMD) that allow efficient interaction with human tetherin. However, despite these adaptive changes, most N-Vpus still antagonize human tetherin only poorly and fail to down-modulate CD4, the natural killer (NK) cell ligand NTB-A as well as the lipid-antigen presenting protein CD1d. These functional deficiencies were mapped to amino acid changes in the cytoplasmic domain that disrupt putative adaptor protein binding sites and an otherwise highly conserved ßTrCP-binding DSGxxS motif. As a consequence, N-Vpus exhibited aberrant intracellular localization and/or failed to recruit the ubiquitin-ligase complex to induce tetherin degradation. The only exception was the Vpu of a group N strain recently discovered in France, but originally acquired in Togo, which contained intact cytoplasmic motifs and counteracted tetherin as effectively as the Vpus of pandemic HIV-1 M strains. These results indicate that HIV-1 group N Vpu is under strong host-specific selection pressure and that the acquisition of effective tetherin antagonism may lead to the emergence of viral variants with increased transmission fitness.

Microbiology: Murder as a solution to promiscuity?

Strain-specific pili enable Vibrio cholerae bacteria to adhere to each other and form aggregates in liquid culture. A new study focuses on strains with less specific, promiscuous pili and suggests a role for contact-dependent bacterial killing in shaping the composition of these aggregates.

Evolution of E. coli in a mouse model of inflammatory bowel disease leads to a disease-specific bacterial genotype and trade-offs with clinical relevance.

Inflammatory bowel disease (IBD) is a persistent inflammatory condition that affects the gastrointestinal tract and presents significant challenges in its management and treatment. Despite the knowledge that within-host bacterial evolution occurs in the intestine, the disease has rarely been studied from an evolutionary perspective. In this study, we aimed to investigate the evolution of resident bacteria during intestinal inflammation and whether- and how disease-related bacterial genetic changes may present trade-offs with potential therapeutic importance. Here, we perform an in vivo evolution experiment of E. coli in a gnotobiotic mouse model of IBD, followed by multiomic analyses to identify disease-specific genetic and phenotypic changes in bacteria that evolved in an inflamed versus a non-inflamed control environment. Our results demonstrate distinct evolutionary changes in E. coli specific to inflammation, including a single nucleotide variant that independently reached high frequency in all inflamed mice. Using ex vivo fitness assays, we find that these changes are associated with a higher fitness in an inflamed environment compared to isolates derived from non-inflamed mice. Further, using large-scale phenotypic assays, we show that bacterial adaptation to inflammation results in clinically relevant phenotypes, which intriguingly include collateral sensitivity to antibiotics. Bacterial evolution in an inflamed gut yields specific genetic and phenotypic signatures. These results may serve as a basis for developing novel evolution-informed treatment approaches for patients with intestinal inflammation.

VasH is a transcriptional regulator of the type VI secretion system functional in endemic and pandemic Vibrio cholerae.

The gram-negative bacterium Vibrio cholerae is the etiological agent of cholera, a disease characterized by the release of high volumes of watery diarrhea. Many medically important proteobacteria, including V. cholerae, carry one or multiple copies of the gene cluster that encodes the bacterial type VI secretion system (T6SS) to confer virulence or interspecies competitiveness. Structural similarity and sequence homology between components of the T6SS and the cell-puncturing device of T4 bacteriophage suggest that the T6SS functions as a molecular syringe to inject effector molecules into prokaryotic and eukaryotic target cells. Although our understanding of how the structural T6SS apparatus assembles is developing, little is known about how this system is regulated. Here, we report on the contribution of the activator of the alternative sigma factor 54, VasH, as a global regulator of the V. cholerae T6SS. Using bioinformatics and mutational analyses, we identified domains of the VasH polypeptide that are essential for its ability to initiate transcription of T6SS genes and established a universal role for VasH in endemic and pandemic V. cholerae strains.

Draft Genome Sequences of 13 Vibrio cholerae Strains from the Rio Grande Delta.

Vibrio cholerae is the etiologic agent of cholera, an acute and often fatal diarrheal disease that affects millions globally. We report the draft genome sequences of 13 non-O1/O139 V. cholerae strains isolated from the Rio Grande Delta in Texas. These genomes will aid future analyses of environmental serovars.

Type VI secretion system mutations reduced competitive fitness of classical Vibrio cholerae biotype.

The gram-negative bacterium Vibrio cholerae is the causative agent of the diarrhoeal disease cholera and is responsible for seven recorded pandemics. Several factors are postulated to have led to the decline of 6th pandemic classical strains and the rise of El Tor biotype V. cholerae, establishing the current 7th pandemic. We investigated the ability of classical V. cholerae of the 2nd and 6th pandemics to engage their type six secretion system (T6SS) in microbial competition against non-pandemic and 7th pandemic strains. We report that classical V. cholerae underwent sequential mutations in T6SS genetic determinants that initially exposed 2nd pandemic strains to microbial attack by non-pandemic strains and subsequently caused 6th pandemic strains to become vulnerable to El Tor biotype V. cholerae intraspecific competition. The chronology of these T6SS-debilitating mutations agrees with the decline of 6th pandemic classical strains and the emergence of 7th pandemic El Tor V. cholerae.

Pandemic Vibrio cholerae shuts down site-specific recombination to retain an interbacterial defence mechanism.

Vibrio cholerae is an aquatic microbe that can be divided into three subtypes: harmless environmental strains, localised pathogenic strains, and pandemic strains causing global cholera outbreaks. Each type has a contact-dependent type VI secretion system (T6SS) that kills neighbouring competitors by translocating unique toxic effector proteins. Pandemic isolates possess identical effectors, indicating that T6SS effectors may affect pandemicity. Here, we show that one of the T6SS gene clusters (Aux3) exists in two states: a mobile, prophage-like element in a small subset of environmental strains, and a truncated Aux3 unique to and conserved in pandemic isolates. Environmental Aux3 can be readily excised from and integrated into the genome via site-specific recombination, whereas pandemic Aux3 recombination is reduced. Our data suggest that environmental Aux3 acquisition conferred increased competitive fitness to pre-pandemic V. cholerae, leading to grounding of the element in the chromosome and propagation throughout the pandemic clade.

The evolution of tit-for-tat in bacteria via the type VI secretion system.

Tit-for-tat is a familiar principle from animal behavior: individuals respond in kind to being helped or harmed by others. Remarkably some bacteria appear to display tit-for-tat behavior, but how this evolved is not understood. Here we combine evolutionary game theory with agent-based modelling of bacterial tit-for-tat, whereby cells stab rivals with poisoned needles (the type VI secretion system) after being stabbed themselves. Our modelling shows tit-for-tat retaliation is a surprisingly poor evolutionary strategy, because tit-for-tat cells lack the first-strike advantage of preemptive attackers. However, if cells retaliate strongly and fire back multiple times, we find that reciprocation is highly effective. We test our predictions by competing Pseudomonas aeruginosa (a tit-for-tat species) with Vibrio cholerae (random-firing), revealing that P. aeruginosa does indeed fire multiple times per incoming attack. Our work suggests bacterial competition has led to a particular form of reciprocation, where the principle is that of strong retaliation, or 'tits-for-tat'.

Evolutionary Approaches to Combat Antibiotic Resistance: Opportunities and Challenges for Precision Medicine.

The rise of antimicrobial resistance (AMR) in bacterial pathogens is acknowledged by the WHO as a major global health crisis. It is estimated that in 2050 annually up to 10 million people will die from infections with drug resistant pathogens if no efficient countermeasures are implemented. Evolution of pathogens lies at the core of this crisis, which enables rapid adaptation to the selective pressures imposed by antimicrobial usage in both medical treatment and agriculture, consequently promoting the spread of resistance genes or alleles in bacterial populations. Approaches developed in the field of Evolutionary Medicine attempt to exploit evolutionary insight into these adaptive processes, with the aim to improve diagnostics and the sustainability of antimicrobial therapy. Here, we review the concept of evolutionary trade-offs in the development of AMR as well as new therapeutic approaches and their impact on host-microbiome-pathogen interactions. We further discuss the possible translation of evolution-informed treatments into clinical practice, considering both the rapid cure of the individual patients and the prevention of AMR.

Adaptor Proteins of Type VI Secretion System Effectors.

Bacteria use the type VI secretion system (T6SS) to kill neighboring cells. One key feature of the T6SS is the secretion of diverse effectors. Here, we discuss six publications that describe three superfamilies of T6SS proteins, each dedicated to mediate the secretion of cognate effectors.

T6SS intraspecific competition orchestrates Vibrio cholerae genotypic diversity.

Vibrio cholerae is a diverse species that inhabits a wide range of environments from copepods in brackish water to the intestines of humans. In order to remain competitive, V. cholerae uses the versatile type-VI secretion system (T6SS) to secrete anti-prokaryotic and anti-eukaryotic effectors. In addition to competing with other bacterial species, V. cholerae strains also compete with one another. Some strains are able to coexist, and are referred to as belonging to the same compatibility group. Challenged by diverse competitors in various environments, different V. choleare strains secrete different combination of effectors - presumably to best suit their niche. Interestingly, all pandemic V. cholerae strains encode the same three effectors. In addition to the diversity displayed in the encoded effectors, the regulation of V. cholerae also differs between strains. Two main layers of regulation appear to exist. One strategy connects T6SS activity with behavior that is suited to fighting eukaryotic cells, while the other is linked with natural competence - the ability of the bacterium to acquire and incorporate extracellular DNA. This relationship between bacterial killing and natural competence is potentially a source of diversification for V. cholerae as it has been shown to incorporate the DNA of cells recently killed through T6SS activity. It is through this process that we hypothesize the transfer of virulence factors, including T6SS effector modules, to happen. Switching of T6SS effectors has the potential to change the range of competitors V. cholerae can kill and to newly define which strains V. cholerae can co-exist with, two important parameters for survival in diverse environments.

Sequential displacement of Type VI Secretion System effector genes leads to evolution of diverse immunity gene arrays in Vibrio cholerae.

Type VI secretion systems (T6SS) enable bacteria to engage neighboring cells in contact-dependent competition. In Vibrio cholerae, three chromosomal clusters each encode a pair of effector and immunity genes downstream of those encoding the T6SS structural machinery for effector delivery. Different combinations of effector-immunity proteins lead to competition between strains of V. cholerae, which are thought to be protected only from the toxicity of their own effectors. Screening of all publically available V. cholerae genomes showed that numerous strains possess long arrays of orphan immunity genes encoded in the 3' region of their T6SS clusters. Phylogenetic analysis reveals that these genes are highly similar to those found in the effector-immunity pairs of other strains, indicating acquisition by horizontal gene transfer. Extensive genomic comparisons also suggest that successive addition of effector-immunity gene pairs replaces ancestral effectors, yet retains the cognate immunity genes. The retention of old immunity genes perhaps provides protection against nearby kin bacteria in which the old effector was not replaced. This mechanism, combined with frequent homologous recombination, is likely responsible for the high diversity of T6SS effector-immunity gene profiles observed for V. cholerae and closely related species.