An Engineered Synthetic Biologic Protects Against Clostridium difficile Infection.

Morbidity and mortality attributed to Clostridium difficile infection (CDI) have increased over the past 20 years. Currently, antibiotics are the only US FDA-approved treatment for primary C. difficile infection, and these are, ironically, associated with disease relapse and the threat of burgeoning drug resistance. We previously showed that non-toxin virulence factors play key roles in CDI, and that colonization factors are critical for disease. Specifically, a C. difficile adhesin, Surface Layer Protein A (SlpA) is a major contributor to host cell attachment. In this work, we engineered Syn-LAB 2.0 and Syn-LAB 2.1, two synthetic biologic agents derived from lactic acid bacteria, to stably and constitutively express a host-cell binding fragment of the C. difficile adhesin SlpA on their cell-surface. Both agents harbor conditional suicide plasmids expressing a codon-optimized chimera of the lactic acid bacterium's cell-wall anchoring surface-protein domain, fused to the conserved, highly adherent, host-cell-binding domain of C. difficile SlpA. Both agents also incorporate engineered biocontrol, obviating the need for any antibiotic selection. Syn-LAB 2.0 and Syn-LAB 2.1 possess positive biophysical and in vivo properties compared with their parental antecedents in that they robustly and constitutively display the SlpA chimera on their cell surface, potentiate human intestinal epithelial barrier function in vitro, are safe, tolerable and palatable to Golden Syrian hamsters and neonatal piglets at high daily doses, and are detectable in animal feces within 24 h of dosing, confirming robust colonization. In combination, the engineered strains also delay (in fixed doses) or prevent (when continuously administered) death of infected hamsters upon challenge with high doses of virulent C. difficile. Finally, fixed-dose Syn-LAB ameliorates diarrhea in a non-lethal model of neonatal piglet enteritis. Taken together, our findings suggest that the two synthetic biologics may be effectively employed as non-antibiotic interventions for CDI.

Secretion Systems and Secreted Proteins in Gram-Negative Entomopathogenic Bacteria: Their Roles in Insect Virulence and Beyond.

Many Gram-negative bacteria have evolved insect pathogenic lifestyles. In all cases, the ability to cause disease in insects involves specific bacterial proteins exported either to the surface, the extracellular environment, or the cytoplasm of the host cell. They also have several distinct mechanisms for secreting such proteins. In this review, we summarize the major protein secretion systems and discuss examples of secreted proteins that contribute to the virulence of a variety of Gram-negative entomopathogenic bacteria, including Photorhabdus, Xenorhabdus, Serratia, Yersinia, and Pseudomonas species. We also briefly summarize two classes of exported protein complexes, the PVC-like elements, and the Tc toxin complexes that were first described in entomopathogenic bacteria.

Variable virulence phenotype of Xenorhabdus bovienii (γ-Proteobacteria: Enterobacteriaceae) in the absence of their vector hosts.

Xenorhabdus bovienii bacteria have a dual lifestyle: they are mutualistic symbionts to many species of Steinernema nematodes and are pathogens to a wide array of insects. Previous studies have shown that virulence of X.bovienii-Steinernema spp. pairs decreases when the nematodes associate with non-cognate bacterial strains. However, the virulence of the X. bovienii strains alone has not been fully investigated. In this study, we characterized the virulence of nine X. bovienii strains in Galleria mellonella and Spodoptera littoralis and performed a comparative genomic analysis to correlate observed phenotypes with strain genotypes. Two X. bovienii strains were found to be highly virulent against the tested insect hosts, while three strains displayed attenuated insect virulence. Comparative genomic analyses revealed the presence of several clusters present only in virulent strains, including a predicted type VI secretion system (T6SS). We performed intra-species-competition assays, and showed that the virulent T6SS+ strains generally outcompeted the less virulent T6SS- strains. Thus, we speculate that the T6SS in X. bovienii may be another addition to the arsenal of antibacterial mechanisms expressed by these bacteria in an insect, where it could potentially play three key roles: (1) competition against the insect host microbiota; (2) protection of the insect cadaver from necrotrophic microbial competitors; and (3) outcompeting other Xenorhabdus species and/or strains when co-infections occur.

Clostridium difficile clinical isolates exhibit variable susceptibility and proteome alterations upon exposure to mammalian cationic antimicrobial peptides.

Clostridium difficile is a leading cause of hospital-acquired bacterial infections in the United States, and the increased incidence of recurrent C. difficile infections is particularly problematic. The molecular mechanisms of C. difficile colonization, including its ability to evade host innate immune responses, is poorly understood. We hypothesized that epidemic-associated C. difficile clinical isolates would exhibit increased resistance to mammalian, gut-associated, cationic antimicrobial peptides such as the cathelicidin LL-37. Standardized susceptibility tests as well as comparative proteomic analyses revealed that C. difficile strains varied in their responses to LL-37, with epidemic-associated 027 ribotype isolates displaying greater resistance. Further, exposure of C. difficile strains to sub-lethal concentrations of LL-37 resulted in increased resistance to subsequent peptide challenge, suggesting the presence of inducible resistance mechanisms. Correspondingly, LL-37 exposure altered the C. difficile proteome, with marked changes in abundance of cell wall biosynthesis proteins, surface layer proteins, ABC transporters and lysine metabolism pathway components. Taken together, these results suggest that innate immune avoidance mechanisms could facilitate robust colonization by C. difficile.

Clostridium difficile infection: toxins and non-toxin virulence factors, and their contributions to disease establishment and host response.

Clostridium difficile infection is the leading cause of antibiotic- and healthcare-associated diarrhea, and its containment and treatment imposes a significant financial burden, estimated to be over $3 billion in the USA alone. Since the year 2000, CDI epidemics/outbreaks have occurred in North America, Europe and Asia. These outbreaks have been variously associated with, or attributed to, the emergence of Clostridium difficile strains with increased virulence, an increase in resistance to commonly used antimicrobials such as the fluoroquinolones, or host susceptibilities, including the use of gastric acid suppressants, to name a few. Efforts to elucidate C. difficile pathogenic mechanisms have been hampered by a lack of molecular tools, manipulatable animal models, and genetic intractability of clinical C. difficile isolates. However, in the past 5 y, painstaking efforts have resulted in the unraveling of multiple C. difficile virulence-associated pathways and mechanisms. We have recently reviewed the disease, its associated risk factors, transmission and interventions (Viswanathan, Gut Microbes 2010). This article summarizes genetics, non-toxin virulence factors, and host-cell biology associated with C. difficile pathogenesis as of 2011, and highlights those findings/factors that may be of interest as future intervention targets.