James T. Summersgill

Distribution of Legionella Species and Serogroups Isolated by Culture in Patients with Sporadic Community-Acquired Legionellosis: An International Collaborative Survey

This international collaborative survey identified culture-confirmed legionellosis in 508 patients with sporadic community-acquired legionellosis. Legionella pneumophila constituted 91.5% of the isolates. Serogroup 1 was the predominant serogroup (84.2%), and serogroups 2-13 (7.4%) accounted for the remaining serogroups. The Legionella species most commonly isolated were L. longbeachae (3.9%) and L. bozemanii (2.4%), followed by L. micdadei, L. dumoffii, L. feeleii, L. wadsworthii, and L. anisa (2.2% combined). L. longbeachae constituted 30.4% of the community-acquired Legionella isolates in Australia and New Zealand.

Chlamydial Persistence beyond the Biphasic Paradigm

The chlamydiae are an evolutionarily distinct group of eubacteria sharing an obligate intracellular lifestyle and a unique developmental cycle that has been well characterized under favorable cell culture conditions. This cycle begins when infectious, metabolically inert elementary bodies (EB)

Inhibition of Chlamydia pneumoniae Replication in HEp-2 Cells by Interferon-γ: Role of Tryptophan Catabolism

Interferon-gamma (IFN-gamma) induces tryptophan catabolism in HEp-2 cells, possibly via stimulation of host cell indoleamine-2,3-dioxygenase activity, in a dose-dependent (12.5-1600 U/mL) fashion after 24 h, resulting in a 99% conversion to its metabolites at 1600 U/mL. Replication of Chlamydia pneumoniae isolates A-03 and BAL-16 was inhibited in HEp-2 cells following treatment with 50 and 100 U/mL IFN-gamma, respectively; however, addition of excess L-tryptophan (200 microg/mL) to monolayers infected with C. pneumoniae resulted in unrestricted growth of both isolates up to 1600 U/mL IFN-gamma. C. pneumoniae could be recovered from IFN-gamma-treated monolayers, indicating the potential for this bacterium to undergo an altered life cycle, in vitro, analogous to that described in detail for Chlamydia trachomatis. The ability of C. pneumoniae to persist in host tissue despite an immunologic response would be an important attribute in order to cause or exacerbate chronic infections.

Proteomic Analysis of Differentially Expressed Chlamydia pneumoniae Genes during Persistent Infection of HEp-2 Cells

ABSTRACT Recent data have shown that the respiratory pathogen Chlamydia pneumoniae expresses an altered gene transcription profile during gamma interferon (IFN-γ)-induced persistent infection in vitro. In the present study, we examined, by proteomics, expression of C. pneumoniae proteins labeled intracellularly with [35S]methionine/cysteine under normal conditions or IFN-γ-mediated persistence. The identity of differentially expressed proteins during persistent infection was determined by matching spots to those of proteins identified in C. pneumoniae elementary bodies by matrix-assisted laser desorption ionization mass spectrometry. Upon treatment with 50 U of IFN-γ per ml, a marked upregulation of major outer membrane protein (MOMP), heat shock protein 60 (Hsp-60/GroEL), and proteins with functions in DNA replication (GyrA), transcription (RpoA, PnP), translation (Rrf), glycolysis (PgK, GlgP), and type III secretion (SctN) was observed at 24 h of infection. In contrast, no significant decreases in bacterial protein expression were found in C. pneumoniae-infected cells due to IFN-γ treatment. Upregulation of C. pneumoniae proteins involved in diverse functions during persistent infection may allow the organism to resist the inhibitory effects of IFN-γ while retaining basic functions. Future studies should examine the differential expression of chlamydial proteins during the developmental cycle under IFN-γ pressure to obtain a finer representation of the gene products involved in establishing persistence.

Characterization of Chlamydia pneumoniae persistence in HEp-2 cells treated with gamma interferon.

ABSTRACT Infection with Chlamydia pneumoniae has been implicated as a potential risk factor for atherosclerosis. This study demonstrated the effects of gamma interferon (IFN-γ)-mediated indoleamine 2,3-dioxygenase activity on C. pneumoniaepersistence in HEp-2 cells, inclusion morphology, and ultrastructure.C. pneumoniae replication showed a dose-dependent decrease when treated with increasing concentrations of IFN-γ and a phenotypic switch resulting in a decrease in typical inclusions with an increase in smaller, less-dense atypical inclusions. Ultrastructural analysis of IFN-γ-treated C. pneumoniae revealed atypical inclusions containing large reticulatate-like aberrant bodies with no evidence of redifferentiation into elementary bodies.

Killing of Legionella pneumophila by nitric oxide in gamma-interferon-activated macrophages.

The role of nitric oxide (NO) radicals in killing the intracellular bacterial pathogen Legionella pneumophila (Lp) was examined in infected macrophages. Murine (RAW 264.7) and human (HL‐60) cell monolayers were treated with 100 U/mlγ‐interferon (IFN) and cocultured with Lp in the presence and absence of NGMMA, a specific inhibitor of NO production. Viable Lp in IFN‐treated RAW 264.7 cells decreased from 3.8 to 0.7 ± 0.12 log CFU/ml after 24 h incubation, whereas in IFN + NGMMA‐treated RAW 264.7 cells, viable Lp persisted at 2.2 ± 0.2 log CFU/ml after 24 h. This increased survival corresponded with an inhibition of NO production (5.65 ± 2.99 μM with NGMMA vs. 58.6 ± 5.36 μM without NGMMA). Viable Lp were susceptible to killing, in a dose‐dependent fashion, by 0, 2.5, and 5.0 mM sodium nitroprusside, a source of NO radicals. IFN‐treated RAW 264.7 cells also had significantly decreased levels of intracellular iron (below assay limit) when compared to IFN + NGMMA‐treated cells (72.0 ± 0.78 % of control). Normally permissive HL‐60 cells treated with IFN were bacteriostatic rather than bactericidal, and NO production was not detected above background. Thus, NO radicals play a critical role in the bactericidal activity against Lp by IFN‐treated RAW 264.7 cells, but the absence of NO production limits IFN‐treated HL‐60 cells to bac‐ teriostasis.

Inhibition of Chlamydia pneumoniae replication in human aortic smooth muscle cells by gamma interferon-induced indoleamine 2, 3-dioxygenase activity

ABSTRACT Infection with Chlamydia pneumoniae, a human respiratory pathogen, has been implicated as a potential risk factor in atherosclerosis, possibly because the pathogen can exist in a persistent form similar to that described for Chlamydia trachomatis. The present study investigated whether gamma interferon (IFN-γ) can induce indoleamine 2,3-dioxygenase (IDO) activity in aortic smooth muscle cells, leading to a marked inhibition of C. pneumoniae growth. Our data indicate a stimulation of IDO mRNA expression and dose-dependent enzymatic activity following IFN-γ treatment. IDO-mediated increase in tryptophan catabolism resulted in a dose-dependent marked inhibition of C. pneumoniae replication.

Requirement for NF-κB in Transcriptional Activation of Monocyte Chemotactic Protein 1 by Chlamydia pneumoniae in Human Endothelial Cells

ABSTRACT Infection with Chlamydia pneumoniae, a causative agent of acute and chronic respiratory diseases, has recently been implicated as a potential risk factor in atherosclerosis. Atherosclerotic lesions are characterized by monocyte infiltration, which may be regulated by the chemokine monocyte chemotactic protein 1 (MCP-1). We have previously shown that C. pneumoniae infection stimulates MCP-1 production in human endothelial cells, an event which may be specific to this species of Chlamydia, sinceChlamydia trachomatis infection fails to induce this response. To examine the underlying mechanisms by which C. pneumoniae infection induces MCP-1 production in endothelial cells, the present study investigated the role of transcription factor NF-κB in MCP-1 mRNA expression. Human umbilical vein endothelial cells (HUVEC) were infected with the coronary isolate C. pneumoniae A-03 or with C. trachomatis L2, and MCP-1 mRNA expression was assessed after different periods of infection by reverse transcription-PCR. Expression of MCP-1 mRNA in C. pneumoniae-infected HUVEC was significantly elevated as early as 1 h postinfection and increased dramatically by 12 and 24 h compared to baseline controls. Nuclear translocation of NF-κB occurred by 30 min of infection, as determined by electrophoretic mobility shift assays and immunofluorescence staining. Treatment ofC. pneumoniae-infected HUVEC with parthenolide, a specific inhibitor of NF-κB activation, suppressed MCP-1 mRNA expression. In contrast, infection with C. trachomatis L2 did not induce MCP-1 mRNA in infected HUVEC and failed to activate NF-κB. Results from this study demonstrate a requirement for NF-κB activation in stimulation of MCP-1 gene expression by C. pneumoniae in human endothelial cells. Furthermore, the data suggest that, within the genus Chlamydia, functionally distinct signaling pathways leading to NF-κB activation are utilized by C. pneumoniaein endothelial cells during infection.

Immunoproteomic Identification and Serological Responses to Novel Chlamydia pneumoniae Antigens That Are Associated with Persistent C. pneumoniae Infections

The controversial discussion about the role of Chlamydia pneumoniae in atherosclerosis cannot be solved without a reliable diagnosis that allows discrimination between past and persistent infections. Using a proteomic approach and immunoblotting with human sera, we identified 31 major C. pneumoniae Ags originating from 27 different C. pneumoniae proteins. More than half of the proteins represent Chlamydia Ags not described previously. Using a comparative analysis of spot reactivity Pmp6, OMP2, GroEL, DnaK, RpoA, EF-Tu, as well as CpB0704 and CpB0837, were found to be immunodominant. The comparison of Ab-response patterns of sera from subjects with and without evidence for persisting C. pneumoniae, determined by multiple PCR analysis of PBMC and vasculatory samples, resulted in differential reactivity for 12 proteins, which is not reflected by reactivity of the sera in the microimmunofluorescence test, the current gold standard for serodiagnosis. Although reactivity of sera from PCR-positive donors was increased toward RpoA, MOMP, YscC, Pmp10, PorB, Pmp21, GroEL, and Cpaf, the reactivity toward YscL, Rho, LCrE, and CpB0837 was decreased, reflecting the altered protein expression of persisting C. pneumoniae in vitro. Our data provide the first evidence of a unique Ab-response pattern associated with persistent C. pneumoniae infections, which is a prerequisite for the serological determination of persistently infected patients.

Protein Expression Profiles of Chlamydia pneumoniae in Models of Persistence versus Those of Heat Shock Stress Response

ABSTRACT Chlamydia pneumoniae is an obligate intracellular pathogen that causes both acute and chronic human disease. Several in vitro models of chlamydial persistence have been established to mimic chlamydial persistence in vivo. We determined the expression patterns of 52 C. pneumoniae proteins, representing nine functional subgroups, from the gamma interferon (IFN-γ) treatment (primarily tryptophan limitation) and iron limitation (IL) models of persistence compared to those following heat shock (HS) at 42°C. Protein expression patterns of C. pneumoniae persistence indicates a strong stress component, as evidenced by the upregulation of proteins involved in protein folding, assembly, and modification. However, it is clearly more than just a stress response. In IFN persistence, but not IL or HS, amino acid and/or nucleotide biosynthesis proteins were found to be significantly upregulated. In contrast, proteins involved in the biosynthesis of cofactors, cellular processes, energy metabolism, transcription, and translation showed an increased in expression in only the IL model of persistence. These data represent the most extensive protein expression study of C. pneumoniae comparing the chlamydial heat shock stress response to two models of persistence and identifying the common and unique protein level responses during persistence.