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ABSTRACTThis study demonstrates a strict temporal requirement for a virulence determinant of the Lyme disease spirochete Borrelia burgdorferiduring a unique point in its natural infection cycle, which alternates between ticks and small mammals. OspC is a major surface protein produced by B. burgdorferiwhen infected ticks feed but whose synthesis decreases after transmission to a mammalian host. We have previously shown that spirochetes lacking OspC are competent to replicate in and migrate to the salivary glands of the tick vector but do not infect mice. Here we assessed the timing of the requirement for OspC by using an ospCmutant complemented with an unstable copy of the ospCgene and show that B. burgdorferi's requirement for OspC is specific to the mammal and limited to a critical early stage of mammalian infection. By using this unique system, we found that most bacterial reisolates from mice persistently infected with the initially complemented ospCmutant strain no longer carried the wild-type copy of ospC. Such spirochetes were acquired by feeding ticks and migrated to the tick salivary glands during subsequent feeding. Despite normal behavior in ticks, these ospCmutant spirochetes did not infect naive mice. ospCmutant spirochetes from persistently infected mice also failed to infect naive mice by tissue transplantation. We conclude that OspC is indispensable for establishing infection by B. burgdorferiin mammals but is not required at any other point of the mouse-tick infection cycle.

ABSTRACTWe previously demonstrated that outer surface protein C (OspC) of Borrelia burgdorferiis essential for establishing mammalian infection. However, the role of OspC in mammalian infection is unknown. Here, we report experiments designed to distinguish between two models of OspC function in the mammalian host: (i) OspC fulfills an essential physiological role for growth and host adaptation or (ii) OspC provides a protective role for evasion of components of the innate immune response. We found that a B. burgdorferi ospCmutant, previously demonstrated to be noninfectious in both immunocompetent and SCID mice, could survive in the relatively immune-privileged environment of dialysis membrane chambers implanted within the peritoneum of a rat. The ospCmutant also adapts to the mammalian environment, as determined by the protein profiles of the chamber-cultivated spirochetes. Therefore, OspC does not appear to provide a physiological function for the survival of B. burgdorferiwithin the mammalian host. The second model, evasion of the innate immune system, was tested by assessing the infectivity of the ospCmutant in mice deficient for myeloid differentiation protein 88 (MyD88). Recent studies have shown that B. burgdorferiis prevented from reaching high cell numbers in the mammalian host by MyD88-dependent signaling pathways. The ospCmutant was incapable of infecting MyD88-deficient mice, suggesting that the role of OspC cannot be related solely to evasion of MyD88-mediated innate immunity. These results reiterate the importance of OspC in mammalian infection and eliminate simple models of function for this enigmatic protein.

ABSTRACTBeta2-toxin, encoded by cpb2, is implicated in the pathogenesis of Clostridium perfringensenteritis. However, cpb2genes from nonporcine C. perfringensisolates were not always expressed, at least in vitro. Nucleotide sequencing identified atypical cpb2genes with 70.2 to 70.7% DNA identity to previously identified (consensus) cpb2. Atypical beta2-toxin displayed 62.3% identity and 80.4% similarity to consensus beta2-toxin. No porcine type C isolates (n= 16) and only 3.3% of porcine type A isolates (n= 60) carried atypical cpb2genes. However, 88.5% of nonporcine isolates carried atypical cpb2(n= 78), but beta2-toxin was not expressed. Almost half of the nonporcine consensus cpb2genes (44.4%) carried a frameshift mutation (n= 9), resulting in an absence of beta2-toxin expression. These findings strengthen the role of beta2-toxin in the pathogenesis of enteritis in neonatal pigs. However, the identification of apparently nonexpressed, atypical cpb2genes raises the question of whether this protein plays the same role in enteritis in other animal species.

Chitobiose is the dimer subunit of chitin, a component of tick cuticle and peritrophic matrix, which is not found in mammals. The Borrelia burgdorferi chbC gene is required for the use of chitobiose as a source of the essential nutrient N-acetyl glucosamine during growth in vitro. In order to investigate the role of chitobiose transport in the infectious cycle, we constructed isogenic chbC mutant and wild-type strains in an infectious B. burgdorferi background and confirmed that the mutants were defective in chitobiose utilization. The defect in the mutants was shown to be in chitobiose transport, consistent with the predicted function of the ChbC protein as the membrane component of a phosphotransferase transporter for chitobiose. We then tested whether this locus is also required for any stage of the experimental mouse-tick infectious cycle. We found that both wild-type and mutant bacteria successfully infect both mice and ticks and are transmitted between the two hosts. These results demonstrate that B. burgdorferi growth in vivo is independent of chitobiose transport, even in an environmental niche in which the sugar is likely to be present.

ABSTRACTAlthough Clostridium perfringensis recognized as an important cause of clostridial enteric diseases, only limited knowledge exists concerning the association of particular C. perfringenstoxinotypes (type A to E) with gastrointestinal (GI) diseases in domestic animals. Some C. perfringensisolates also produce the newly discovered beta2-toxin (CPB2). Recent epidemiological studies suggested that C. perfringensisolates carrying the gene encoding CPB2 (cpb2) are strongly associated with clostridial GI diseases in domestic animals, including necrotic enteritis in piglets and typhlocolitis in horses. These putative relationships, obtained by PCR genotyping, were tested in the present study by further genotyping and phenotyping of 29 cpb2-positive C. perfringensisolates from pigs with GI disease (pig GI disease isolates). PCR and restriction fragment length polymorphism analysis reconfirmed the presence of cpb2gene sequences in all the disease isolates included in the study. Furthermore, genotyping by pulsed-field gel electrophoresis analyses showed that the pig GI disease isolates included in this study all carry a plasmid cpb2gene, yet no clonal relationships were detected between the cpb2-positive pig GI disease isolates surveyed. Finally, CPB2-specific Western blotting demonstrated CPB2 expression by all of the cpb2-positive isolates surveyed. The CPB2 proteins made by five of these pig GI disease isolates were shown to have the same deduced amino acid sequences as the biologically active CPB2 protein made by the original type C isolate, CWC245. Collectively, our present results support a significant association between CPB2-positive C. perfringensisolates and diarrhea in piglets.

ABSTRACTSeveral Clostridium perfringensgenotype E isolates, all associated with hemorrhagic enteritis of neonatal calves, were identified by multiplex PCR. These genotype E isolates were demonstrated to express α and ι toxins, but, despite carrying sequences for the gene (cpe) encoding C. perfringensenterotoxin (CPE), were unable to express CPE. These silent cpesequences were shown to be highly conserved among type E isolates. However, relative to the functionalcpegene of type A isolates, these silent type Ecpesequences were found to contain nine nonsense and two frameshift mutations and to lack the initiation codon, promoters, and ribosome binding site. The type E animal enteritis isolates carrying these silent cpesequences do not appear to be clonally related, and their silent type E cpesequences are always located, near the ι toxin genes, on episomal DNA. These findings suggest that the highly conserved, silent cpesequences present in most or all type E isolates may have resulted from the recent horizontal transfer of an episome, which also carries ι toxin genes, to several different type A C. perfringensisolates.