Identification of novel toxins associated with the extracellular contractile injection system using machine learning.

Secretion systems play a crucial role in microbe-microbe or host-microbe interactions. Among these systems, the extracellular contractile injection system (eCIS) is a unique bacterial and archaeal extracellular secretion system that injects protein toxins into target organisms. However, the specific proteins that eCISs inject into target cells and their functions remain largely unknown. Here, we developed a machine learning classifier to identify eCIS-associated toxins (EATs). The classifier combines genetic and biochemical features to identify EATs. We also developed a score for the eCIS N-terminal signal peptide to predict EAT loading. Using the classifier we classified 2,194 genes from 950 genomes as putative EATs. We validated four new EATs, EAT14-17, showing toxicity in bacterial and eukaryotic cells, and identified residues of their respective active sites that are critical for toxicity. Finally, we show that EAT14 inhibits mitogenic signaling in human cells. Our study provides insights into the diversity and functions of EATs and demonstrates machine learning capability of identifying novel toxins. The toxins can be employed in various applications dependently or independently of eCIS.

Hierarchical determinants in cytotoxic necrotizing factor (CNF) toxins driving Rho G-protein deamidation versus transglutamination.

The cytotoxic necrotizing factor (CNF) family of AB-type bacterial protein toxins catalyze two types of modification on their Rho GTPase substrates: deamidation and transglutamination. It has been established that E. coli CNF1 and its close homolog proteins catalyze primarily deamidation and Bordetella dermonecrotic toxin (DNT) catalyzes primarily transglutamination. The rapidly expanding microbial genome sequencing data have revealed that there are at least 13 full-length variants of CNF1 homologs. CNFx from E. coli strain GN02091 is the most distant from all other members of the CNF family with 50%-55% sequence identity at the protein level and 0.45-0.52 nucleotide substitutions per site at the DNA level. CNFx modifies RhoA, Rac1, and Cdc42, and like CNF1, activates downstream SRE-dependent mitogenic signaling pathways in human HEK293T cells, but at a 1,000-fold higher EC50 value. Unlike other previously characterized CNF toxins, CNFx modifies Rho proteins primarily through transglutamination, as evidenced by gel-shift assay and confirmed by MALDI mass spectral analysis, when coexpressed with Rho-protein substrates in E. coli BL21 cells or through direct treatment of HEK293T cells. A comparison of CNF1 and CNFx sequences identified two critical active-site residues corresponding to positions 832 and 862 in CNF1. Reciprocal site-specific mutations at these residues in each toxin revealed hierarchical rules that define the preference for deamidase versus a transglutaminase activity in CNFs. An additional unique Cys residue at the C-terminus of CNFx was also discovered to be critical for retarding cargo delivery.IMPORTANCECytotoxic necrotizing factor (CNF) toxins not only play important virulence roles in pathogenic E. coli and other bacterial pathogens, but CNF-like genes have also been found in an expanding number of genomes from clinical isolates. Harnessing the power of evolutionary relationships among the CNF toxins enabled the deciphering of the hierarchical active-site determinants that define whether they modify their Rho GTPase substrates through deamidation or transglutamination. With our finding that a distant CNF variant (CNFx) unlike other known CNFs predominantly transglutaminates its Rho GTPase substrates, the paradigm of "CNFs deamidate and DNTs transglutaminate" could finally be attributed to two critical amino acid residues within the active site other than the previously identified catalytic Cys-His dyad residues. The significance of our approach and research findings is that they can be applied to deciphering enzyme reaction determinants and substrate specificities for other bacterial proteins in the development of precision therapeutic strategies.

Vascular Endothelial Growth Factor A Contributes to Increased Mammalian Respiratory Epithelial Permeability Induced by Pasteurella multocida Infection.

Pasteurella multocida infection can cause significant zoonotic respiratory problems in both humans and animals, but little is known about the mechanisms used by P. multocida to invade and cross the mammalian respiratory barrier. In this study, we investigated the influence of P. multocida infection on the dysfunction of the respiratory epithelial barrier. In vivo tests in mouse infection models demonstrated that P. multocida infection significantly increased epithelial permeability and increased the expression of vascular endothelial growth factor A (VEGFA) and endothelial nitric oxide synthase (eNOS) in murine tracheae and lungs. In murine lung epithelial cell (MLE-12) models, P. multocida infection decreased the expression of tight junctions (ZO-1) and adherens junctions (ß-catenin and E-cadherin) proteins but induced the activation of hypoxia-inducible factor 1α (HIF-1α) and VEGFA signaling. When the expression of HIF-1α is suppressed, the induction of VEGFA and ZO-1 expression by P. multocida infection is decreased. We also found that intervention of HIF-1α and VEGFA signaling affected infection outcomes caused by respiratory bacteria in mouse models. Most importantly, we demonstrate that P. multocida infection increases the permeability of human respiratory epithelial cells and that this process is associated with the activation of HIF-1α and VEGFA signaling and likely contributes to the pathogenesis of P. multocida infection in humans. IMPORTANCE The mammalian respiratory epithelium forms the first line of defense against infections with P. multocida, an important zoonotic respiratory pathogen. In this study, we found that P. multocida infection increased respiratory epithelial permeability and promoted the induction of the HIF-1α-VEGFA axis in both mouse and murine cell models. Similar findings were also demonstrated in human respiratory epithelial cells. The results from this study provide important knowledge about the pathogenesis of P. multocida causing infections in both animals and humans.

Two Bordetella bronchiseptica attenuated vaccine candidates confer protection against lethal challenge with B. Bronchiseptica and Pasteurella multocida toxin in mouse models.

Dermonecrotic toxin (DNT) is an important bacterial virulence factor produced by the zoonotic pathogens Bordetella bronchiseptica and Pasteurella multocida. This study aims to explore the possibility of expressing different fragments of P. multocida toxin (PMT) in the chromosome of attenuated B. bronchiseptica to generate single-component mucosal vaccine candidates. To achieve this, a 954-bp fragment (basepairs 301 âˆ¼ 1254) of the B. bronchiseptica aroA gene was replaced with an N-terminal, 930-bp fragment (basepairs 1-930; PMTN) or a C-terminal, 900-bp fragment (base pairs 2959 âˆ¼ 3858; PMTC) of the PMT encoding gene toxA. The resulting strains, denoted as Bb-PMTN or Bb-PMTC, expressed PMTN and PMTC, as evidenced by ELISA using polyclonal against full-length of PMT. Phenotypical analyses revealed that Bb-PMTN and Bb-PMTC grew much slower than wild type strains in tryptic soy broth. These strains also displayed significantly decreased 161-fold-virulence compared to the wildtype strains in mouse models. Intranasal immunization of Bb-PMTN and Bb-PMTC in mice induced high levels of antibodies against B. bronchiseptica and PMT, as well as IFN-γ and IL-10 in mouse sera, and most importantly, high titers of sIgA in mouse lungs. Vaccination with these two engineering strains provided 100% protection of mice against lethal challenge with B. bronchiseptica and 80%∼100% protection against lethal challenge with PMT, with Bb-PMTN exhibiting 1.25-fold greater immunogenic efficacy over Bb-PMTC. This study highlights the use of B. bronchiseptica attenuated strains as live mucosal vectors to deliver heterologous antigens.

Insertion-trigger residues differentially modulate endosomal escape by cytotoxic necrotizing factor toxins.

The cellular specificity, potency, and modular nature of bacterial protein toxins enable their application for targeted cytosolic delivery of therapeutic cargo. Efficient endosomal escape is a critical step in the design of bacterial toxin-inspired drug delivery (BTIDD) vehicles to avoid lysosomal degradation and promote optimal cargo delivery. The cytotoxic necrotizing factor (CNF) family of modular toxins represents a useful model for investigating cargo-delivery mechanisms due to the availability of many homologs with high sequence identity, their flexibility in swapping domains, and their differential activity profiles. Previously, we found that CNFy is more sensitive to endosomal acidification inhibitors than CNF1 and CNF2. Here, we report that CNF3 is even less sensitive than CNF1/2. We identified two amino acid residues within the putative translocation domain (E374 and E412 in CNFy, Q373 and S411 in CNF3) that differentiate between these two toxins. Swapping these corresponding residues in each toxin changed the sensitivity to endosomal acidification and efficiency of cargo-delivery to be more similar to the other toxin. Results suggested that trafficking to the more acidic late endosome is required for cargo delivery by CNFy but not CNF3. This model was supported by results from toxin treatment of cells in the presence of NH4Cl, which blocks endosomal acidification, and of small-molecule inhibitors EGA, which blocks trafficking to late endosomes, and ABMA, which blocks endosomal escape and trafficking to the lysosomal degradative pathway. These findings suggest that it is possible to fine-tune endosomal escape and cytosolic cargo delivery efficiency in designing BTIDD platforms.

Recovery of microbial community profile information hidden in chimeric sequence reads.

The next frontier in the field of microbiome studies is identification of all microbes present in the microbiome and accurate determination of their abundance such that microbiome profiles can serve as reliable assessments of health or disease status. PCR-based 16S rRNA gene sequencing and metagenome shotgun sequencing technologies are the prevailing approaches used in microbiome analyses. Each poses a number of technical challenges associated with PCR amplification, sample availability, and cost of processing and analysis. In general, results from these two approaches rarely agree completely with each other. Here, we compare these methods utilizing a set of vaginal swab and lavage specimens from a cohort of 42 pregnant women collected for a pilot study exploring the effect of the vaginal microbiome on preterm birth. We generated the microbial community profiles from the sequencing reads of the V3V4 and V4V5 regions of the 16S rRNA gene in the vaginal swab and lavage samples. For a subset of the vaginal samples from 12 subjects, we also performed metagenomic shotgun sequencing analysis and compared the results obtained from the PCR-based sequencing methods. Our findings suggest that sample composition and complexity, particularly at the species level, are major factors that must be considered when analyzing and interpreting microbiome data. Our approach to sequence analysis includes consideration of chimeric reads, by using our chimera-counting BlastBin program, and enables recovery of microbial content information generated during PCR-based sequencing methods, such that the microbial profiles more closely resemble those obtained from metagenomic read-based approaches.

Development of an Online Tool for Pasteurella multocida Genotyping and Genotypes of Pasteurella multocida From Different Hosts.

Pasteurella multocida is a versatile zoonotic pathogen. Multiple systems have been applied to type P. multocida from different diseases in different hosts. Recently, we found that assigning P. multocida strains by combining their capsular, lipopolysaccharide, and MLST genotypes (marked as capsular: lipopolysaccharide: MLST genotype) could help address the biological characteristics of P. multocida circulation in different hosts. However, there is still lack of a rapid and efficient tool to diagnose P. multocida according to this system. Here, we developed an intelligent genotyping platform PmGT for P. multocida strains according to their whole genome sequences using the web 2.0 technologies. By using PmGT, we determined capsular genotypes, LPS genotypes, and MLST genotypes as well as the main virulence factor genes (VFGs) of P. multocida isolates from different host species based on their whole genome sequences published on NCBI. The results revealed a closer association between the genotypes and pasteurellosis rather than between genotypes and host species. With the advent of high-quality, inexpensive DNA sequencing, PmGT represents a more efficient tool for P. multocida diagnosis in both epidemiological studies and clinical settings.

Traditional Human Populations and Nonhuman Primates Show Parallel Gut Microbiome Adaptations to Analogous Ecological Conditions.

Compared with urban-industrial populations, small-scale human communities worldwide share a significant number of gut microbiome traits with nonhuman primates. This overlap is thought to be driven by analogous dietary triggers; however, the ecological and functional bases of this similarity are not fully understood. To start addressing this issue, fecal metagenomes of BaAka hunter-gatherers and traditional Bantu agriculturalists from the Central African Republic were profiled and compared with those of a sympatric western lowland gorilla group (Gorilla gorilla gorilla) across two seasons of variable dietary intake. Results show that gorilla gut microbiomes shared similar functional traits with each human group, depending on seasonal dietary behavior. Specifically, parallel microbiome traits were observed between hunter-gatherers and gorillas when the latter consumed more structural polysaccharides during dry seasons, while small-scale agriculturalist and gorilla microbiomes showed significant functional overlap when gorillas consumed more seasonal ripe fruit during wet seasons. Notably, dominance of microbial transporters, transduction systems, and gut xenobiotic metabolism was observed in association with traditional agriculture and energy-dense diets in gorillas at the expense of a functional microbiome repertoire capable of metabolizing more complex polysaccharides. Differential abundance of bacterial taxa that typically distinguish traditional from industrialized human populations (e.g., Prevotella spp.) was also recapitulated in the human and gorilla groups studied, possibly reflecting the degree of polysaccharide complexity included in each group's dietary niche. These results show conserved functional gut microbiome adaptations to analogous diets in small-scale human populations and nonhuman primates, highlighting the role of plant dietary polysaccharides and diverse environmental exposures in this convergence.IMPORTANCE The results of this study highlight parallel gut microbiome traits in human and nonhuman primates, depending on subsistence strategy. Although these similarities have been reported before, the functional and ecological bases of this convergence are not fully understood. Here, we show that this parallelism is, in part, likely modulated by the complexity of plant carbohydrates consumed and by exposures to diverse xenobiotics of natural and artificial origin. Furthermore, we discuss how divergence from these parallel microbiome traits is typically associated with adverse health outcomes in human populations living under culturally westernized subsistence patterns. This is important information as we trace the specific dietary and environmental triggers associated with the loss and gain of microbial functions as humans adapt to various dietary niches.

Pasteurella multocida: Genotypes and Genomics.

Pasteurella multocida is a highly versatile pathogen capable of causing infections in a wide range of domestic and wild animals as well as in humans and nonhuman primates. Despite over 135 years of research, the molecular basis for the myriad manifestations of P. multocida pathogenesis and the determinants of P. multocida phylogeny remain poorly defined. The current availability of multiple P. multocida genome sequences now makes it possible to delve into the underlying genetic mechanisms of P. multocida fitness and virulence. Using whole-genome sequences, the genotypes, including the capsular genotypes, lipopolysaccharide (LPS) genotypes, and multilocus sequence types, as well as virulence factor-encoding genes of P. multocida isolates from different clinical presentations can be characterized rapidly and accurately. Putative genetic factors that contribute to virulence, fitness, host specificity, and disease predilection can also be identified through comparative genome analysis of different P. multocida isolates. However, although some knowledge about genotypes, fitness, and pathogenesis has been gained from the recent whole-genome sequencing and comparative analysis studies of P. multocida, there is still a long way to go before we fully understand the pathogenic mechanisms of this important zoonotic pathogen. The quality of several available genome sequences is low, as they are assemblies with relatively low coverage, and genomes of P. multocida isolates from some uncommon host species are still limited or lacking. Here, we review recent advances, as well as continuing knowledge gaps, in our understanding of determinants contributing to virulence, fitness, host specificity, disease predilection, and phylogeny of P. multocida.

Plasticity in the Human Gut Microbiome Defies Evolutionary Constraints.

The gut microbiome of primates, including humans, is reported to closely follow host evolutionary history, with gut microbiome composition being specific to the genetic background of its primate host. However, the comparative models used to date have mainly included a limited set of closely related primates. To further understand the forces that shape the primate gut microbiome, with reference to human populations, we expanded the comparative analysis of variation among gut microbiome compositions and their primate hosts, including 9 different primate species and 4 human groups characterized by a diverse set of subsistence patterns (n = 448 samples). The results show that the taxonomic composition of the human gut microbiome, at the genus level, exhibits increased compositional plasticity. Specifically, we show unexpected similarities between African Old World monkeys that rely on eclectic foraging and human populations engaging in nonindustrial subsistence patterns; these similarities transcend host phylogenetic constraints. Thus, instead of following evolutionary trends that would make their microbiomes more similar to that of conspecifics or more phylogenetically similar apes, gut microbiome composition in humans from nonindustrial populations resembles that of generalist cercopithecine monkeys. We also document that wild cercopithecine monkeys with eclectic diets and humans following nonindustrial subsistence patterns harbor high gut microbiome diversity that is not only higher than that seen in humans engaging in industrialized lifestyles but also higher compared to wild primates that typically consume fiber-rich diets.IMPORTANCE The results of this study indicate a discordance between gut microbiome composition and evolutionary history in primates, calling into question previous notions about host genetic control of the primate gut microbiome. Microbiome similarities between humans consuming nonindustrialized diets and monkeys characterized by subsisting on eclectic, omnivorous diets also raise questions about the ecological and nutritional drivers shaping the human gut microbiome. Moreover, a more detailed understanding of the factors associated with gut microbiome plasticity in primates offers a framework to understand why humans following industrialized lifestyles have deviated from states thought to reflect human evolutionary history. The results also provide perspectives for developing therapeutic dietary manipulations that can reset configurations of the gut microbiome to potentially improve human health.

Gut microbiome composition of wild western lowland gorillas is associated with individual age and sex factors.

OBJECTIVES: Environmental and ecological factors, such as geographic range, anthropogenic pressure, group identity, and feeding behavior are known to influence the gastrointestinal microbiomes of great apes. However, the influence of individual host traits such as age and sex, given specific dietary and social constraints, has been less studied. The objective of this investigation was to determine the associations between an individual's age and sex on the diversity and composition of the gut microbiome in wild western lowland gorillas. MATERIALS AND METHODS: Publicly available 16S rRNA data generated from fecal samples of different groups of Gorilla gorilla gorilla in the Central African Republic were downloaded and bioinformatically processed. The groups analyzed included habituated, partially habituated and unhabituated gorillas, sampled during low fruit (dry, n = 28) and high fruit (wet, n = 82) seasons. Microbial community analyses (alpha and beta diversity and analyses of discriminant taxa), in tandem with network-wide approaches, were used to (a) mine for specific age and sex based differences in gut bacterial community composition and to (b) asses for gut community modularity and bacterial taxa with potential functional roles, in the context of seasonal food variation, and social group affiliation. RESULTS: Both age and sex significantly influenced gut microbiome diversity and composition in wild western lowland gorillas. However, the largest differences were observed between infants and adults in habituated groups and between adults and immature gorillas within all groups, and across dry and wet seasons. Specifically, although adults always showed greater bacterial richness than infants and immature gorillas, network-wide analyses showed higher microbial community complexity and modularity in the infant gorilla gut. Sex-based microbiome differences were not evident among adults, being only detected among immature gorillas. CONCLUSIONS: The results presented point to a dynamic gut microbiome in Gorilla spp., associated with ontogeny and individual development. Of note, the gut microbiomes of breastfeeding infants seemed to reflect early exposure to complex, herbaceous vegetation. Whether increased compositional complexity of the infant gorilla gut microbiome is an adaptive response to an energy-limited diet and an underdeveloped gut needs to be further tested. Overall, age and sex based gut microbiome differences, as shown here, maybe mainly attributed to access to specific feeding sources, and social interactions between individuals within groups.

Comparative Genome Analysis of an Extensively Drug-Resistant Isolate of Avian Sequence Type 167 Escherichia coli Strain Sanji with Novel In Silico Serotype O89b:H9.

Extensive drug resistance (XDR) is an escalating global problem. Escherichia coli strain Sanji was isolated from an outbreak of pheasant colibacillosis in Fujian province, China, in 2011. This strain has XDR properties, exhibiting sensitivity to carbapenems but no other classes of known antibiotics. Whole-genome sequencing revealed a total of 32 known antibiotic resistance genes, many associated with insertion sequence 26 (IS26) elements. These were found on the Sanji chromosome and 2 of its 6 plasmids, pSJ_255 and pSJ_82. The Sanji chromosome also harbors a type 2 secretion system (T2SS), a type 3 secretion system (T3SS), a type 6 secretion system (T6SS), and several putative prophages. Sanji and other ST167 strains have a previously uncharacterized O-antigen (O89b) that is most closely related to serotype O89 as determined on the basis of analysis of the wzm-wzt genes and in silico serotyping. This O89b-antigen gene cluster was also found in the genomes of a few other pathogenic sequence type 617 (ST617) and ST10 complex strains. A time-scaled phylogeny inferred from comparative single nucleotide variant analysis indicated that development of these O89b-containing lineages emerged about 30 years ago. Comparative sequence analysis revealed that the core genome of Sanji is nearly identical to that of several recently sequenced strains of pathogenic XDR E. coli belonging to the ST167 group. Comparison of the mobile elements among the different ST167 genomes revealed that each genome carries a distinct set of multidrug resistance genes on different types of plasmids, indicating that there are multiple paths toward the emergence of XDR in E. coli. IMPORTANCE E. coli strain Sanji is the first sequenced and analyzed genome of the recently emerged pathogenic XDR strains with sequence type ST167 and novel in silico serotype O89b:H9. Comparison of the genomes of Sanji with other ST167 strains revealed distinct sets of different plasmids, mobile IS elements, and antibiotic resistance genes in each genome, indicating that there exist multiple paths toward achieving XDR. The emergence of these pathogenic ST167 E. coli strains with diverse XDR capabilities highlights the difficulty of preventing or mitigating the development of XDR properties in bacteria and points to the importance of better understanding of the shared underlying virulence mechanisms and physiology of pathogenic bacteria.

Relationships Between Gastrointestinal Parasite Infections and the Fecal Microbiome in Free-Ranging Western Lowland Gorillas.

Relationships between gastrointestinal parasites (GIPs) and the gastrointestinal microbiome (GIM) are widely discussed topics across mammalian species due to their possible impact on the host's health. GIPs may change the environment determining alterations in GIM composition. We evaluated the associations between GIP infections and fecal microbiome composition in two habituated and two unhabituated groups of wild western lowland gorillas (Gorilla g. gorilla) from Dzanga Sangha Protected Areas, Central African Republic. We examined 43 fecal samples for GIPs and quantified strongylid nematodes. We characterized fecal microbiome composition through 454 pyrosequencing of the V1-V3 region of the bacterial 16S rRNA gene. Entamoeba spp. infections were associated with significant differences in abundances of bacterial taxa that likely play important roles in nutrition and metabolism for the host, besides being characteristic members of the gorilla gut microbiome. We did not observe any relationships between relative abundances of several bacterial taxa and strongylid egg counts. Based on our findings, we suggest that there is a significant relationship between fecal microbiome and Entamoeba infection in wild gorillas. This study contributes to the overall knowledge about factors involved in modulating GIM communities in great apes.

CNF1-like deamidase domains: common Lego bricks among cancer-promoting immunomodulatory bacterial virulence factors.

Alterations of the cellular proteome over time due to spontaneous or toxin-mediated enzymatic deamidation of glutamine (Gln) and asparagine (Asn) residues contribute to bacterial infection and might represent a source of aging-related diseases. Here, we put into perspective what is known about the mode of action of the CNF1 toxin from pathogenic Escherichia coli, a paradigm of bacterial deamidases that activate Rho GTPases, to illustrate the importance of determining whether exposure to these factors are risk factors in the etiology age-related diseases, such as cancer. In particular, through in silico analysis of the distribution of the CNF1-like deamidase active site Gly-Cys-(Xaa)n-His sequence motif in bacterial genomes, we unveil the wide distribution of the super-family of CNF-like toxins and CNF-like deamidase domains among members of the Enterobacteriacae and in association with a large variety of toxin delivery systems. We extent our discussion with recent findings concerning cellular systems that control activated Rac1 GTPase stability and provide protection against cancer. These findings point to the urgency for developing holistic approaches toward personalized medicine that include monitoring for asymptomatic carriage of pathogenic toxin-producing bacteria and that ultimately might lead to improved public health and increased lifespans.

Cytosolic Delivery of Multidomain Cargos by the N Terminus of Pasteurella multocida Toxin.

The zoonotic pathogen Pasteurella multocida produces a 146-kDa modular toxin (PMT) that enters host cells and manipulates intracellular signaling through action on its Gα protein targets. The N terminus of PMT (PMT-N) mediates cellular uptake through receptor-mediated endocytosis, followed by the delivery of the C-terminal catalytic domain from acidic endosomes into the cytosol. The putative native cargo of PMT consists of a 710-residue polypeptide with three distinct modular subdomains (C1-C2-C3), where C1 contains a membrane localization domain (MLD), C2 has an as-yet-undefined function, and C3 catalyzes the deamidation of a specific active-site glutamine residue in Gα protein targets. However, whether the three cargo subdomains are delivered intact or undergo further proteolytic processing during or after translocation from the late endosome is unclear. Here, we demonstrate that PMT-N mediates the delivery of its native C-terminal cargo as a single polypeptide, corresponding to C1-C2-C3, including the MLD, with no evidence of cleavage between subdomains. We show that PMT-N also delivers nonnative green fluorescent protein (GFP) cargo into the cytosol, further supporting that the receptor-binding and translocation functions reside within PMT-N. Our findings further show that PMT-N can deliver C1-C2 alone but that the presence of C1-C2 is important for the cytosolic delivery of the catalytic C3 subdomain by PMT-N. In addition, we further refine the minimum C3 domain required for intracellular activity as comprising residues 1105 to 1278. These findings reinforce that PMT-N serves as the cytosolic delivery vehicle for C-terminal cargo and demonstrate that its native cargo is delivered intact as C1-C2-C3.

Modular domain swapping among the bacterial cytotoxic necrotizing factor (CNF) family for efficient cargo delivery into mammalian cells.

Modular AB-type bacterial protein toxins target mammalian host cells with high specificity and deliver their toxic cargo into the cytosol. Hence, these toxins are being explored as agents for targeted cytosolic delivery in biomedical and research applications. The cytotoxic necrotizing factor (CNF) family is unique among these toxins in that their homologous sequences are found in a wide array of bacteria, and their activity domains are packaged in various delivery systems. Here, to study how CNF cargo and delivery modules can be assembled for efficient cytosolic delivery, we generated chimeric toxins by swapping functional domains among CNF1, CNF2, CNF3, and CNFy. Chimeras with a CNFy delivery vehicle were more stably expressed, but were less efficient at cargo delivery into HEK293-T cells. We also found that CNFy cargo is the most universally compatible and that CNF3 delivery vehicle is the most flexible and efficient at delivering cargo. These findings suggest that domains within proteins can be swapped and accommodate each other for efficient function and that an individual domain could be engineered for compatibility with multiple partner domains. We anticipate that our insights could help inform chemical biology approaches to develop toxin-based cargo-delivery platforms for cytosolic cargo delivery of therapeutics or molecular probes into mammalian cells.

Impact of stress on the gut microbiome of free-ranging western lowland gorillas.

Exposure to stressors can negatively impact the mammalian gastrointestinal microbiome (GIM). Here, we used 454 pyrosequencing of 16S rRNA bacterial gene amplicons to evaluate the impact of physiological stress, as evidenced by faecal glucocorticoid metabolites (FGCM; ng/g), on the GIM composition of free-ranging western lowland gorillas (Gorilla gorilla gorilla). Although we found no relationship between GIM alpha diversity (H) and FGCM levels, we observed a significant relationship between the relative abundances of particular bacterial taxa and FGCM levels. Specifically, members of the family Anaerolineaceae (ρ=0.4, FDR q=0.01), genus Clostridium cluster XIVb (ρ=0.35, FDR q=0.02) and genus Oscillibacter (ρ=0.35, FDR q=0.02) were positively correlated with FGCM levels. Thus, while exposure to stressors appears to be associated with minor changes in the gorilla GIM, the consequences of these changes are unknown. Our results may have implications for conservation biology as well as for our overall understanding of factors influencing the non-human primate GIM.

The membrane localization domains of two distinct bacterial toxins form a 4-helix-bundle in solution.

Membrane localization domain (MLD) was first proposed for a 4-helix-bundle motif in the crystal structure of the C1 domain of Pasteurella multocida toxin (PMT). This structure motif is also found in the crystal structures of several clostridial glycosylating toxins (TcdA, TcdB, TcsL, and TcnA). The Ras/Rap1-specific endopeptidase (RRSP) module of the multifunctional autoprocessing repeats-in-toxins (MARTX) toxin produced by Vibrio vulnificus has sequence homology to the C1-C2 domains of PMT, including a putative MLD. We have determined the solution structure for the MLDs in PMT and in RRSP using solution state NMR. We conclude that the MLDs in these two toxins assume a 4-helix-bundle structure in solution.