John D. Klena

Bacterial polysaccharide synthesis and gene nomenclature

Gene nomenclature for bacterial surface polysaccharides is complicated by the large number of structures and genes. We propose a scheme applicable to all species that distinguishes different classes of genes, provides a single name for all genes of a given function and greatly facilitates comparative studies.

Secretion of Virulence Proteins from Campylobacter jejuni Is Dependent on a Functional Flagellar Export Apparatus

Campylobacter jejuni, a gram-negative motile bacterium, secretes a set of proteins termed the Campylobacter invasion antigens (Cia proteins). The purpose of this study was to determine whether the flagellar apparatus serves as the export apparatus for the Cia proteins. Mutations were generated in five genes encoding three structural components of the flagella, the flagellar basal body (flgB and flgC), hook (flgE2), and filament (flaA and flaB) genes, as well as in genes whose products are essential for flagellar protein export (flhB and fliI). While mutations that affected filament assembly were found to be nonmotile (Mot-) and did not secrete Cia proteins (S-), a flaA (flaB+) filament mutant was found to be nonmotile but Cia protein secretion competent (Mot-, S+). Complementation of a flaA flaB double mutant with a shuttle plasmid harboring either the flaA or flaB gene restored Cia protein secretion, suggesting that Cia export requires at least one of the two filament proteins. Infection of INT 407 human intestinal cells with the C. jejuni mutants revealed that maximal invasion of the epithelial cells required motile bacteria that are secretion competent. Collectively, these data suggest that the C. jejuni Cia proteins are secreted from the flagellar export apparatus.

A Family Cluster of Infections by a Newly Recognized Bunyavirus in Eastern China, 2007: Further Evidence of Person-to-Person Transmission

BACKGROUND Seven persons in one family living in eastern China developed fever and thrombocytopenia during May 2007, but the initial investigation failed to identify an infectious etiology. In December 2009, a novel bunyavirus (designated severe fever with thrombocytopenia syndrome bunyavirus [SFTSV]) was identified as the cause of illness in patients with similar clinical manifestations in China. We reexamined this family cluster for SFTSV infection. METHODS We analyzed epidemiological and clinical data for the index patient and 6 secondary patients. We tested stored blood specimens from the 6 secondary patients using real time reverse transcription polymerase chain reaction (RT-PCR), viral culture, genetic sequencing, micro-neutralization assay (MNA), and indirect immunofluorescence assay (IFA). RESULTS An 80-year-old woman with fever, leucopenia, and thrombocytopenia died on 27 April 2007. Between 3 and 7 May 2007, another 6 patients from her family were admitted to a local county hospital with fever and other similar symptoms. Serum specimens collected in 2007 from these 6 patients were positive for SFTS viral RNA through RT-PCR and for antibody to SFTSV through MNA and IFA. SFTSV was isolated from 1 preserved serum specimen. The only shared characteristic between secondary patients was personal contact with the index patient; none reported exposure to suspected animals or vectors. CONCLUSIONS Clinical and laboratory evidence confirmed that the patients of fever and thrombocytopenia occurring in a family cluster in eastern China in 2007 were caused by a newly recognized bunyavirus, SFTSV. Epidemiological investigation strongly suggests that infection of secondary patients was transmitted to family members by personal contact.

Characterization of Toxigenic Vibrio cholerae from Haiti, 2010-2011

A virulent clone from Africa or southern Asia was likely introduced at a single time point.

Fatal Case of Campylobacter lari Prosthetic Joint Infection and Bacteremia in an Immunocompetent Patient

ABSTRACT Campylobacter lari is an infrequent cause of intestinal and extraintestinal infection in humans. We report a case of C. lari prosthetic joint infection and bacteremia in an 81-year-old immunocompetent man. The infection was associated with septic shock and fatal outcome. C. lari may cause severe disease, even in an immunocompetent host.

Characterization of Genetically Matched Isolates of Campylobacter jejuni Reveals that Mutations in Genes Involved in Flagellar Biosynthesis Alter the Organism's Virulence Potential

ABSTRACT Phenotypic and genotypic evidence suggests that not all Campylobacter jejuni isolates are pathogenic for humans. We hypothesized that differences in gene content or gene expression alter the degree of pathogenicity of C. jejuni isolates. A C. jejuni isolate (Turkey) recovered from a turkey and a second C. jejuni isolate (CS) recovered from a chicken differed in their degrees of in vitro and in vivo virulence. The C. jejuni Turkey isolate invaded INT 407 human epithelial cells and secreted the Cia (Campylobacter invasion antigen) proteins, while the C. jejuni CS isolate was noninvasive for human epithelial cells and did not secrete the Cia proteins. Newborn piglets inoculated with the C. jejuni Turkey isolate developed more severe clinical signs of campylobacteriosis than piglets inoculated with the C. jejuni CS isolate. Additional work revealed that flagellin was not expressed in the C. jejuni CS isolate. Microarray and real-time reverse transcription-PCR analyses revealed that all flagellar class II genes were significantly downregulated in the C. jejuni CS isolate compared to the C. jejuni Turkey isolate. Finally, nucleotide sequencing of the flgR gene revealed the presence of a single residue that was different in the FlgR proteins of the C. jejuni Turkey and CS isolates. Complementation of the C. jejuni CS isolate with a wild-type copy of the flgR gene restored the isolates motility. Collectively, these findings support the hypothesis that critical differences in gene content or gene expression can alter the pathogenic potential of C. jejuni isolates.

Plasminogen Activator Pla of Yersinia pestis Utilizes Murine DEC-205 (CD205) as a Receptor to Promote Dissemination

Yersinia pestis, a Gram-negative bacterium that causes bubonic and pneumonic plague, is able to rapidly disseminate to other parts of its mammalian hosts. Y. pestis expresses plasminogen activator (PLA) on its surface, which has been suggested to play a role in bacterial dissemination. It has been speculated that Y. pestis hijacks antigen-presenting cells, such as macrophages (MΦs) and dendritic cells, to be delivered to lymph nodes to initiate dissemination and infection. Both alveolar MΦs and pulmonary dendritic cells express a C-type lectin receptor, DEC-205 (CD205), which mediates antigen uptake and presentation. However, no ligand has been identified for DEC-205. In this study, we show that the invasion of alveolar MΦsby Y. pestis depends both in vitro and in vivo on the expression of PLA. DEC-205-expressing MΦs and transfectants, but not their negative counterparts, phagocytosed PLA-expressing Y. pestis and Escherichia coli K12 more efficiently than PLA-negative controls. The interactions between PLA-expressing bacteria and DEC-205-expressing transfectants or alveolar MΦs could be inhibited by an anti-DEC-205 antibody. Importantly, the blockage of the PLA-DEC-205 interaction reduced the dissemination of Y. pestis in mice. In conclusion, murine DEC-205 is a receptor for PLA of Y. pestis, and this host-pathogen interaction appears to play a key role in promoting bacterial dissemination.

Laboratory-Based Surveillance of Nontyphoidal Salmonella Infections in China

Foodborne infections are an important public health problem in China. In 2008, we conducted surveillance for laboratory-confirmed nontyphoidal Salmonella to monitor trends for this infection in China and to build capacity for rapid detection and response to foodborne outbreaks. Salmonella isolates from patients with diarrhea were sent from hospitals to local public health laboratories for confirmation, serotyping, and antimicrobial susceptibility testing. A total of 126 hospitals in 44 cities and counties from 8 provinces provided isolates and epidemiologic data for analysis. Of 23,140 stool specimens submitted to clinical laboratories, 662 (3%) grew Salmonella enterica. Salmonella were most commonly detected between April and October. The median age of infected patients was 27 years; 34% of infections occurred in patients <5 years old. Of the 662 isolates, we found 73 serotypes, of which serotype Enteritidis (31%) and serotype Typhimurium (26%) were the most common. The prevalence of resistance was high for clinically important antimicrobial agents, including ampicillin (41%) and ciprofloxacin (6%). More than 60% of isolates, including 35% of all Typhimurium, were resistant to three or more antimicrobial agents. In this first multiprovince surveillance report of laboratory-confirmed Salmonella infections in China, we found that Enteritidis and Typhimurium are the most common serotypes and that efforts to reduce antimicrobial resistance among Salmonella in China are needed. Although no outbreaks were detected using this system, efforts to improve this systems capacity to do so are underway.

Role of N-Acetylglucosamine within Core Lipopolysaccharide of Several Species of Gram-Negative Bacteria in Targeting the DC-SIGN (CD209)

Our recent studies have shown that the dendritic cell-specific ICAM nonintegrin CD209 (DC-SIGN) specifically binds to the core LPS of Escherichia coli K12 (E. coli), promoting bacterial adherence and phagocytosis. In this current study, we attempted to map the sites within the core LPS that are directly involved in LPS-DC-SIGN interaction. We took advantage of four sets of well-defined core LPS mutants, which are derived from E. coli, Salmonella enterica serovar Typhimurium, Neisseria gonorrhoeae, and Haemophilus ducreyi and determined interaction of each of these four sets with DC-SIGN. Our results demonstrated that N-acetylglucosamine (GlcNAc) sugar residues within the core LPS in these bacteria play an essential role in targeting the DC-SIGN receptor. Our results also imply that DC-SIGN is an innate immune receptor and the interaction of bacterial core LPS and DC-SIGN may represent a primeval interaction between Gram-negative bacteria and host phagocytic cells.

The rfaE gene from Escherichia coli encodes a bifunctional protein involved in biosynthesis of the lipopolysaccharide core precursor ADP-L- glycero-D-manno-heptose

The intermediate steps in the biosynthesis of the ADP-L-glycero-D-manno-heptose precursor of inner core lipopolysaccharide (LPS) are not yet elucidated. We isolated a mini-Tn10 insertion that confers a heptoseless LPS phenotype in the chromosome of Escherichia coli K-12. The mutation was in a gene homologous to the previously reported rfaE gene from Haemophilus influenzae. The E. coli rfaE gene was cloned into an expression vector, and an in vitro transcription-translation experiment revealed a polypeptide of approximately 55 kDa in mass. Comparisons of the predicted amino acid sequence with other proteins in the database showed the presence of two clearly separate domains. Domain I (amino acids 1 to 318) shared structural features with members of the ribokinase family, while Domain II (amino acids 344 to 477) had conserved features of the cytidylyltransferase superfamily that includes the aut gene product of Ralstonia eutrophus. Each domain was expressed individually, demonstrating that only Domain I could complement the rfaE::Tn10 mutation in E. coli, as well as the rfaE543 mutation of Salmonella enterica SL1102. DNA sequencing of the rfaE543 gene revealed that Domain I had one amino acid substitution and a 12-bp in-frame deletion resulting in the loss of four amino acids, while Domain II remained intact. We also demonstrated that the aut::Tn5 mutation in R. eutrophus is associated with heptoseless LPS, and this phenotype was restored following the introduction of a plasmid expressing the E. coli Domain II. Thus, both domains of rfaE are functionally different and genetically separable confirming that the encoded protein is bifunctional. We propose that Domain I is involved in the synthesis of D-glycero-D-manno-heptose 1-phosphate, whereas Domain II catalyzes the ADP transfer to form ADP-D-glycero-D-manno-heptose.