Cell free DNA from respiratory pathogens is detectable in the blood plasma of Cystic Fibrosis patients.

Diagnostically informative microbial cell-free DNA (cfDNA) can be detected from blood plasma during fulminant infections such as sepsis. However, the potential for DNA from airway pathogens to enter the circulation of cystic fibrosis (CF) patients during chronic infective states has not yet been evaluated. We assessed whether patient blood contained measurable quantities of cfDNA from CF respiratory microorganisms by sequencing plasma from 21 individuals with CF recruited from outpatient clinics and 12 healthy controls. To account for possible contamination with exogenous microbial nucleic acids, statistical significance of microbe-derived read counts from CF patients was determined relative to the healthy control population. In aggregate, relative abundance of microbial cfDNA was nearly an order of magnitude higher in CF patients than in healthy subjects (p = 8.0×10-3). 15 of 21 (71%) CF patients demonstrated cfDNA from one or more relevant organisms. In contrast, none of the healthy subjects evidenced significant microbial cfDNA for any of the organisms examined. Concordance of cfDNA with standard microbiological culture of contemporaneously collected patient sputum was variable. Our findings provide evidence that cfDNA from respiratory pathogens are present in the bloodstream of most CF patients, which could potentially be exploited for the purposes of noninvasive clinical diagnosis.

Evasion of Toll-like receptor 5 by flagellated bacteria.

Toll-like receptor 5 (TLR5) recognizes an evolutionarily conserved site on bacterial flagellin that is required for flagellar filament assembly and motility. The alpha and epsilon Proteobacteria, including the important human pathogens Campylobacter jejuni, Helicobacter pylori, and Bartonella bacilliformis, require flagellar motility to efficiently infect mammalian hosts. In this study, we demonstrate that these bacteria make flagellin molecules that are not recognized by TLR5. We map the site responsible for TLR5 evasion to amino acids 89-96 of the N-terminal D1 domain, which is centrally positioned within the previously defined TLR5 recognition site. Salmonella flagellin is strongly recognized by TLR5, but mutating residues 89-96 to the corresponding H. pylori flaA sequence abolishes TLR5 recognition and also destroys bacterial motility. To preserve bacterial motility, alpha and epsilon Proteobacteria possess compensatory amino acid changes in other regions of the flagellin molecule, and we engineer a mutant form of Salmonella flagellin that evades TLR5 but retains motility. These results suggest that TLR5 evasion is critical for the survival of this subset of bacteria at mucosal sites in animals and raise the intriguing possibility that flagellin receptors provided the selective force to drive the evolution of these unique subclasses of bacterial flagellins.

FliC-specific CD4+ T cell responses are restricted by bacterial regulation of antigen expression.

Salmonella typhimurium, a facultatively intracellular pathogen, regulates expression of virulence factors in response to distinct environments encountered during the course of infection. We tested the hypothesis that the transition from extra- to intracellular environments during Salmonella infection triggers changes in Ag expression that impose both temporal and spatial limitations on the host T cell response. CD4(+) T cells recovered from Salmonella immune mice were propagated in vitro using Ag derived from bacteria grown in conditions designed to emulate extra- or intracellular environments in vivo. Extracellular phase bacteria supported a dominant T cell response to the flagellar subunit protein FliC, whereas intracellular phase bacteria were unable to support expansion of FliC-specific T cells from populations known to contain T cells with reactivity to this Ag. This result was attributed to bacterial regulation of FliC expression: transcription and protein levels were repressed in bacteria growing in the spleens of infected mice. Furthermore, Salmonella-infected splenocytes taken directly ex vivo stimulated FliC-specific T cell clones only when intracellular FliC expression was artificially up-regulated. Although it has been suggested that a microanatomical separation of immune T cells and infected APC exists in vivo, we demonstrate that intracellular Salmonella can repress FliC expression below the T cell activation threshold. This potentially provides a mechanism for intracellular Salmonella at systemic sites to avoid detection by Ag-specific T cells primed at intestinal sites early in infection.

CD4+ T cells and toll-like receptors recognize Salmonella antigens expressed in bacterial surface organelles.

A better understanding of immunity to infection is revealed from the characteristics of microbial ligands recognized by host immune responses. Murine infection with the intracellular bacterium Salmonella generates CD4+ T cells that specifically recognize Salmonella proteins expressed in bacterial surface organelles such as flagella and membrane vesicles. These natural Salmonella antigens are also ligands for Toll-like receptors (TLRs) or avidly associated with TLR ligands such as lipopolysaccharide (LPS). PhoP/PhoQ, a regulon controlling Salmonella virulence and remodeling of LPS to resist innate immunity, coordinately represses production of surface-exposed antigens recognized by CD4+ T cells and TLRs. These data suggest that genetically coordinated surface modifications may provide a growth advantage for Salmonella in host tissues by limiting both innate and adaptive immune recognition.

Characterization of a novel group of mycobacteria and proposal of Mycobacterium sherrisii sp. nov.

We describe here the characterization of five isolates of Mycobacterium simiae-like organisms representing a novel group based on whole-cell fatty acid analysis and genotypic evaluation. Two of the five isolates in this study, W55 and W58, were previously considered to belong to M. simiae serotype 2. Analysis of cellular fatty acids by gas-liquid chromatography indicated a close clustering of this group, which was well differentiated from the other M. simiae-like species. Molecular characterization was performed by nucleic acid sequencing of the small subunit rRNA gene and the gene encoding the 65-kDa heat shock protein and genomic DNA hybridization. Sequence analysis of the entire 16S rRNA gene showed a unique sequence most closely related to those of M. triplex and M. simiae. The hsp65 partial gene sequence was identical for the five isolates, with 97% identity to the M. simiae type strain. However, qualitative whole genomic DNA hybridization analysis confirmed that this group is genetically distinct from M. simiae and M. triplex. Antimicrobial susceptibilities for this group resemble those of M. simiae and M. lentiflavum. We conclude that this group represents a unique Mycobacterium species for which we propose the name Mycobacterium sherrisii sp. nov.

Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility.

Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and activates host inflammatory responses. In this study, we examine the nature of the TLR5-flagellin interaction. With deletional, insertional and alanine-scanning mutagenesis, we precisely mapped the TLR5 recognition site on flagellin to a cluster of 13 amino acid residues that participate in intermolecular interactions within flagellar protofilaments and that are required for bacterial motility. The recognition site is buried in the flagellar filament, and monomeric flagellin, but not the filamentous molecule, stimulated TLR5. Finally, flagellin coprecipitated with TLR5, indicating close physical interaction between the molecules. These studies demonstrate the exquisite ability of the innate immune system to precisely target a conserved site on flagellin that is essential for bacterial motility.