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.

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.

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.

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.

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.

Selective Membrane Redistribution and Depletion of Gαq-Protein by Pasteurella multocida Toxin.

Pasteurella multocida toxin (PMT), the major virulence factor responsible for zoonotic atrophic rhinitis, is a protein deamidase that activates the alpha subunit of heterotrimeric G proteins. Initial activation of G alpha-q-coupled phospholipase C-beta-1 signaling by PMT is followed by uncoupling of G alpha-q-dependent signaling, causing downregulation of downstream calcium and mitogenic signaling pathways. Here, we show that PMT decreases endogenous and exogenously expressed G alpha-q protein content in host cell plasma membranes and in detergent resistant membrane (DRM) fractions. This membrane depletion of G alpha-q protein was dependent upon the catalytic activity of PMT. Results indicate that PMT-modified G alpha-q redistributes within the host cell membrane from the DRM fraction into the soluble membrane and cytosolic fractions. In contrast, PMT had no affect on G alpha-s or G beta protein levels, which are not substrate targets of PMT. PMT also had no affect on G alpha-11 levels, even though G alpha-11 can serve as a substrate for deamidation by PMT, suggesting that membrane depletion of PMT-modified G-alpha-q has specificity.

Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B.

Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.

Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity.

The paralytic disease botulism is caused by botulinum neurotoxins (BoNT), multi-domain proteins containing a zinc endopeptidase that cleaves the cognate SNARE protein, thereby blocking acetylcholine neurotransmitter release. Antitoxins currently used to treat botulism neutralize circulating BoNT but cannot enter, bind to or neutralize BoNT that has already entered the neuron. The light chain endopeptidase domain (LC) of BoNT serotype A (BoNT/A) was targeted for generation of monoclonal antibodies (mAbs) that could reverse paralysis resulting from intoxication by BoNT/A. Single-chain variable fragment (scFv) libraries from immunized humans and mice were displayed on the surface of yeast, and 19 BoNT/A LC-specific mAbs were isolated by using fluorescence-activated cell sorting (FACS). Affinities of the mAbs for BoNT/A LC ranged from a KD value of 9.0×10-11 M to 3.53×10-8 M (mean KD 5.38×10-9 M and median KD 1.53×10-9 M), as determined by flow cytometry analysis. Eleven mAbs inhibited BoNT/A LC catalytic activity with IC50 values ranging from 8.3 ~73×10-9 M. The fine epitopes of selected mAbs were also mapped by alanine-scanning mutagenesis, revealing that the inhibitory mAbs bound the α-exosite region remote from the BoNT/A LC catalytic center. The results provide mAbs that could prove useful for intracellular reversal of paralysis post-intoxication and further define epitopes that could be targeted by small molecule inhibitors.

Altered active zones, vesicle pools, nerve terminal conductivity, and morphology during experimental MuSK myasthenia gravis.

Recent studies demonstrate reduced motor-nerve function during autoimmune muscle-specific tyrosine kinase (MuSK) myasthenia gravis (MG). To further understand the basis of motor-nerve dysfunction during MuSK-MG, we immunized female C57/B6 mice with purified rat MuSK ectodomain. Nerve-muscle preparations were dissected and neuromuscular junctions (NMJs) studied electrophysiologically, morphologically, and biochemically. While all mice produced antibodies to MuSK, only 40% developed respiratory muscle weakness. In vitro study of respiratory nerve-muscle preparations isolated from these affected mice revealed that 78% of NMJs produced endplate currents (EPCs) with significantly reduced quantal content, although potentiation and depression at 50 Hz remained qualitatively normal. EPC and mEPC amplitude variability indicated significantly reduced number of vesicle-release sites (active zones) and reduced probability of vesicle release. The readily releasable vesicle pool size and the frequency of large amplitude mEPCs also declined. The remaining NMJs had intermittent (4%) or complete (18%) failure of neurotransmitter release in response to 50 Hz nerve stimulation, presumably due to blocked action potential entry into the nerve terminal, which may arise from nerve terminal swelling and thinning. Since MuSK-MG-affected muscles do not express the AChR γ subunit, the observed prolongation of EPC decay time was not due to inactivity-induced expression of embryonic acetylcholine receptor, but rather to reduced catalytic activity of acetylcholinesterase. Muscle protein levels of MuSK did not change. These findings provide novel insight into the pathophysiology of autoimmune MuSK-MG.

Cargo-delivery platforms for targeted delivery of inhibitor cargos against botulism.

Delivering therapeutic cargos to specific cell types in vivo poses many technical challenges. There is currently a plethora of drug leads and therapies against numerous diseases, ranging from small molecule compounds to nucleic acids to peptides to proteins with varying binding or enzymatic functions. Many of these candidate therapies have documented potential for mitigating or reversing disease symptoms, if only a means for gaining access to the intracellular target were available. Recent advances in our understanding of the biology of cellular uptake and transport processes and the mode of action of bacterial protein toxins have accelerated the development of toxin-based cargo-delivery vehicle platforms. This review provides an updated survey of the status of available platforms for targeted delivery of therapeutic cargos, outlining various strategies that have been used to deliver different types of cargo into cells. Particular emphasis is placed on the application of toxin-based approaches, examining critical issues that have hampered realization of post-intoxication antitoxins against botulism.

Primate vaginal microbiomes exhibit species specificity without universal Lactobacillus dominance.

Bacterial communities colonizing the reproductive tracts of primates (including humans) impact the health, survival and fitness of the host, and thereby the evolution of the host species. Despite their importance, we currently have a poor understanding of primate microbiomes. The composition and structure of microbial communities vary considerably depending on the host and environmental factors. We conducted comparative analyses of the primate vaginal microbiome using pyrosequencing of the 16S rRNA genes of a phylogenetically broad range of primates to test for factors affecting the diversity of primate vaginal ecosystems. The nine primate species included: humans (Homo sapiens), yellow baboons (Papio cynocephalus), olive baboons (Papio anubis), lemurs (Propithecus diadema), howler monkeys (Alouatta pigra), red colobus (Piliocolobus rufomitratus), vervets (Chlorocebus aethiops), mangabeys (Cercocebus atys) and chimpanzees (Pan troglodytes). Our results indicated that all primates exhibited host-specific vaginal microbiota and that humans were distinct from other primates in both microbiome composition and diversity. In contrast to the gut microbiome, the vaginal microbiome showed limited congruence with host phylogeny, and neither captivity nor diet elicited substantial effects on the vaginal microbiomes of primates. Permutational multivariate analysis of variance and Wilcoxon tests revealed correlations among vaginal microbiota and host species-specific socioecological factors, particularly related to sexuality, including: female promiscuity, baculum length, gestation time, mating group size and neonatal birth weight. The proportion of unclassified taxa observed in nonhuman primate samples increased with phylogenetic distance from humans, indicative of the existence of previously unrecognized microbial taxa. These findings contribute to our understanding of host-microbe variation and coevolution, microbial biogeography, and disease risk, and have important implications for the use of animal models in studies of human sexual and reproductive diseases.

Determinants of pH-dependent modulation of translocation in dermonecrotic G-protein-deamidating toxins.

Cytotoxic necrotizing factors from E. coli (CNF1, CNF2) and Yersinia (CNFy)share N-terminal sequence similarity with Pasteurella multocida toxin (PMT). This common N-terminal region harbors the receptor-binding and translocation domains that mediate uptake and delivery of the C-terminal catalytic cargo domains into the host cytosol. Subtle variations in the N-terminal ~500 amino acids of CNFs and PMT could allow for selective recognition of cellular receptors and thus, selective target cell specificity. Through studies with cellular inhibitors, we have identified an additional novel function for this region in modulating responses of these toxin proteins to changes in pH during intoxication and delivery of the catalytic cargo domain into the cytosol.

Pasteurella multocida: from zoonosis to cellular microbiology.

In a world where most emerging and reemerging infectious diseases are zoonotic in nature and our contacts with both domestic and wild animals abound, there is growing awareness of the potential for human acquisition of animal diseases. Like other Pasteurellaceae, Pasteurella species are highly prevalent among animal populations, where they are often found as part of the normal microbiota of the oral, nasopharyngeal, and upper respiratory tracts. Many Pasteurella species are opportunistic pathogens that can cause endemic disease and are associated increasingly with epizootic outbreaks. Zoonotic transmission to humans usually occurs through animal bites or contact with nasal secretions, with P. multocida being the most prevalent isolate observed in human infections. Here we review recent comparative genomics and molecular pathogenesis studies that have advanced our understanding of the multiple virulence mechanisms employed by Pasteurella species to establish acute and chronic infections. We also summarize efforts being explored to enhance our ability to rapidly and accurately identify and distinguish among clinical isolates and to control pasteurellosis by improved development of new vaccines and treatment regimens.

A multi-omic systems-based approach reveals metabolic markers of bacterial vaginosis and insight into the disease.

BACKGROUND: Bacterial vaginosis (BV) is the most common vaginal disorder of reproductive-age women. Yet the cause of BV has not been established. To uncover key determinants of BV, we employed a multi-omic, systems-biology approach, including both deep 16S rRNA gene-based sequencing and metabolomics of lavage samples from 36 women. These women varied demographically, behaviorally, and in terms of health status and symptoms. PRINCIPAL FINDINGS: 16S rRNA gene-based community composition profiles reflected Nugent scores, but not Amsel criteria. In contrast, metabolomic profiles were markedly more concordant with Amsel criteria. Metabolomic profiles revealed two distinct symptomatic BV types (SBVI and SBVII) with similar characteristics that indicated disruption of epithelial integrity, but each type was correlated to the presence of different microbial taxa and metabolites, as well as to different host behaviors. The characteristic odor associated with BV was linked to increases in putrescine and cadaverine, which were both linked to Dialister spp. Additional correlations were seen with the presence of discharge, 2-methyl-2-hydroxybutanoic acid, and Mobiluncus spp., and with pain, diethylene glycol and Gardnerella spp. CONCLUSIONS: The results not only provide useful diagnostic biomarkers, but also may ultimately provide much needed insight into the determinants of BV.

Rho/ROCK-dependent inhibition of 3T3-L1 adipogenesis by G-protein-deamidating dermonecrotic toxins: differential regulation of Notch1, Pref1/Dlk1, and ß-catenin signaling.

The dermonecrotic toxins from Pasteurella multocida (PMT), Bordetella (DNT), Escherichia coli (CNF1-3), and Yersinia (CNFY) modulate their G-protein targets through deamidation and/or transglutamination of an active site Gln residue, which results in activation of the G protein and its cognate downstream signaling pathways. Whereas DNT and the CNFs act on small Rho GTPases, PMT acts on the α subunit of heterotrimeric G(q), G(i), and G(12/13) proteins. We previously demonstrated that PMT potently blocks adipogenesis and adipocyte differentiation in a calcineurin-independent manner through downregulation of Notch1 and stabilization of ß-catenin and Pref1/Dlk1, key proteins in signaling pathways strongly linked to cell fate decisions, including fat and bone development. Here, we report that similar to PMT, DNT, and CNF1 completely block adipogenesis and adipocyte differentiation by preventing upregulation of adipocyte markers, PPARγ and C/EBPα, while stabilizing the expression of Pref1/Dlk1 and ß-catenin. We show that the Rho/ROCK inhibitor Y-27632 prevented or reversed these toxin-mediated effects, strongly supporting a role for Rho/ROCK signaling in dermonecrotic toxin-mediated inhibition of adipogenesis and adipocyte differentiation. Toxin treatment was also accompanied by downregulation of Notch1 expression, although this inhibition was independent of Rho/ROCK signaling. We further show that PMT-mediated downregulation of Notch1 expression occurs primarily through G(12/13) signaling. Our results reveal new details of the pathways involved in dermonecrotic toxin action on adipocyte differentiation, and the role of Rho/ROCK signaling in mediating toxin effects on Wnt/ß-catenin and Notch1 signaling, and in particular the role of G(q) and G(12/13) in mediating PMT effects on Rho/ROCK and Notch1 signaling.