Kensuke Shima

Proteome and Transcriptome Analysis of the Virulence Genes Regulated by the VirR/VirS System in Clostridium perfringens

The proteins under the control of the two-component system VirR/VirS in Clostridium perfringens were analyzed by using two-dimensional gel electrophoresis of the culture supernatant from the wild type and the virR mutant. Based on matrix-assisted laser desorption ionization-time of flight/mass spectrometry, seven positively regulated proteins and eight negatively regulated proteins were identified. Transcriptome analysis confirmed that 7 of the 15 proteins were regulated by the VirR/VirS system at the transcriptional level, but the remaining proteins were modified with a VirR/VirS-directed protease at the posttranslation and secretion levels. We purified and characterized the VirR/VirS-directed protease from the culture supernatant and identified it as a kind of clostripain. Because this proteolytic activity was strongly inhibited by leupeptin and antipain, it was concluded that this protease was a member of the family of cysteine proteases of C. perfringens.

An inducible lambdoid prophage encoding cytolethal distending toxin (Cdt-I) and a type III effector protein in enteropathogenic Escherichia coli

Cytolethal distending toxins (CDTs) are inhibitory cyclomodulins, which block eukaryotic cell proliferation and are produced by a diverse group of Gram-negative bacteria, including Escherichia coli strains associated with intestinal and extraintestinal infections. However, the mode of transmission of the toxin gene clusters among diverse bacterial pathogens is unclear. We found that Cdt-I produced by enteropathogenic E. coli strains associated with diarrhea is encoded by a lambdoid prophage, which is inducible and infectious. The genome of Cdt-I converting phage (CDT-1Φ) comprises 47,021 nucleotides with 60 predicted ORFs organized into six genomic regions encoding the head and tail, virulence, integrase, unknown functions, regulation, and lysis. The genomic organization of CDT-1Φ is similar to those of SfV, a serotype-converting phage of Shigella flexneri, and UTI89, a prophage identified in uropathogenic E. coli. Besides the cdtI gene cluster, the virulence region of CDT-1Φ genome contains sequences homologous to a truncated cycle inhibiting factor and a type 3 effector protein. Mutation analysis of susceptible E. coli strain C600 suggested that the outer membrane protein OmpC is a putative receptor for CDT-1Φ. CDT-1Φ genome was also found to integrate into the host bacterial chromosome forming lysogens, which produced biologically active Cdt-I. Furthermore, phage induction appeared to cause enhanced toxigenicity of the E. coli strains carrying lysogenic CDT-1Φ. Our results suggest that CDT-1Φ is the latest member of a growing family of lambdoid phages encoding bacterial cyclomodulins and that the phage may have a role in horizontal transfer of these virulence genes.

Chlamydia pneumoniae infection and Alzheimer’s disease: a connection to remember?

Alzheimer’s disease (AD) is the most common cause of dementia in the elderly, whereby it is customary to distinguish between early familial FAD and late-onset AD (LOAD). The development of LOAD, the most prevalent form of AD, is believed to be a multifactorial process that may also involve infections with bacterial or viral pathogens. After the first report on the presence of Chlamydia pneumoniae (Cpn) in brains of patients with AD appeared in 1998, this bacterium has most often been implicated in AD pathogenesis. However, while some studies demonstrate a clear association between Cpn infection and AD, others have failed to confirm these findings. This might be due to heterogeneity of the specimens analyzed and lack of standardized detection methods. Additionally, non-availability of suitable chlamydial infection models severely hampers research in the field. In this review, we will critically discuss the possible role of Cpn in the pathogenesis of LOAD in light of the available data. We will also present three mutually non-exclusive hypotheses how Cpn might contribute to the pathogenesis of AD.

Fluorescence Lifetime Imaging Unravels C. trachomatis Metabolism and Its Crosstalk with the Host Cell

Chlamydia trachomatis is an obligate intracellular bacterium that alternates between two metabolically different developmental forms. We performed fluorescence lifetime imaging (FLIM) of the metabolic coenzymes, reduced nicotinamide adenine dinucleotides [NAD(P)H], by two-photon microscopy for separate analysis of host and pathogen metabolism during intracellular chlamydial infections. NAD(P)H autofluorescence was detected inside the chlamydial inclusion and showed enhanced signal intensity on the inclusion membrane as demonstrated by the co-localization with the 14-3-3β host cell protein. An increase of the fluorescence lifetime of protein-bound NAD(P)H [τ2-NAD(P)H] inside the chlamydial inclusion strongly correlated with enhanced metabolic activity of chlamydial reticulate bodies during the mid-phase of infection. Inhibition of host cell metabolism that resulted in aberrant intracellular chlamydial inclusion morphology completely abrogated the τ2-NAD(P)H increase inside the chlamydial inclusion. τ2-NAD(P)H also decreased inside chlamydial inclusions when the cells were treated with IFNγ reflecting the reduced metabolism of persistent chlamydiae. Furthermore, a significant increase in τ2-NAD(P)H and a decrease in the relative amount of free NAD(P)H inside the host cell nucleus indicated cellular starvation during intracellular chlamydial infection. Using FLIM analysis by two-photon microscopy we could visualize for the first time metabolic pathogen-host interactions during intracellular Chlamydia trachomatis infections with high spatial and temporal resolution in living cells. Our findings suggest that intracellular chlamydial metabolism is directly linked to cellular NAD(P)H signaling pathways that are involved in host cell survival and longevity.

Prevalence and characteristics of cytolethal distending toxin-producing Escherichia coli from children with diarrhea in Japan

In the present study, we examined the prevalence and characteristics of CTEC among diarrheal children in Japan during a year‐long surveillance study. A PCR‐RFLP assay for the detection and differentiation of five types of E. coli cdtB gene (types I through V) was developed, and 362 stool specimens collected from patients reporting to pediatric departments in two hospitals were analyzed. Of the 35 samples (9.7%) that were positive for the cdtB gene, 21 were positive for cdt‐I, three for cdt‐II, four for cdt‐III, three for cdt‐IV and four samples were positive for cdt‐V, as determined by different molecular techniques. The recovery of CTEC having cdt alleles was a little less, which included 19 with cdt‐I, one cdt‐II, three cdt‐III, three cdt‐IV and four with cdt‐V. Among 30 CTEC strains isolated, the majority of them (43%) belonged to serogroup O2. The other virulence genes such as astA, cnf1, eaeA, cnf2 and bfpA genes were detected in 14 (47%), 11 (37%), four (13%), three (10%) and one (3.3%) strains of CTEC, respectively. However, the other common virulence‐associated genes specific for DEC were not detected in these strains. Interestingly, an untypable cdt gene was detected by PCR‐RFLP in Providencia alcalifaciens. Our data indicate that CTEC may be associated with diarrheal children in Japan and most of them do not belong to a conventional enteropathogenic pathovar and thus differ from strains isolated in developing countries.

When oxygen runs short: the microenvironment drives host–pathogen interactions

Pathogens that colonize or infect the human body have to face varying oxygen concentrations within different organs. Inflammation itself promotes oxygen consumption within affected tissues and creates a low oxygen environment. As a consequence, pathogens and the host immune system have to adapt to rapid changes in oxygen availability. Here we summarize recent findings on the adaptation of pathogens, host defense mechanisms and treatment strategies against intracellular pathogens in a low oxygen environment.

Development of a PCR-Restriction Fragment Length Polymorphism Assay for the Epidemiological Analysis of Shiga Toxin-Producing Escherichia coli

ABSTRACT Six characteristic regions (I to VI) were identified in Shiga toxin 2 (Stx2) phages (T. Sato, T. Shimizu, M. Watarai, M. Kobayashi, S. Kano, T. Hamabata, Y. Takeda, and S. Yamasaki, Gene 309:35-48, 2003). Region V, which is ca. 10 kb in size and is located in the upstream region of the Stx operons, includes the most distinctive region among six Stx phages whose genome sequences have been determined. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay for the epidemiological analysis of Shiga toxin-producing Escherichia coli (STEC) on the basis of the diversity of region V. When region V was amplified by long and accurate-PCR (LA-PCR) with five control E. coli strains carrying six different Stx phages such as E. coli strains C600 (Stx1 phage), C600 (933W phage), C600 (Stx2 phage-I), C600 (Stx2 phage-II), and O157:H7 Sakai strain RIMD0509952 (VT1-Sakai phage and VT2-Sakai phage), an expected size of the band was obtained. Restriction digest of each PCR product with BglI or EcoRV also gave the expected sizes of banding patterns and discriminated the RFLPs of five control strains. When a total of 204 STEC O157 strains were analyzed by LA-PCR, one to three bands whose sizes ranged from 8.2 to 14 kb were obtained. Two STEC O157 strains, however, did not produce any bands. Subsequent restriction digest of the PCR products with BglI or EcoRV differentiated the RFLPs of 202 STEC O157 strains into 24 groups. The RFLP patterns of pulsed-field gel electrophoresis (PFGE) of representative strains of STEC O157 divided into 24 groups were well correlated with those of PCR-RFLP when STEC O157 strains were isolated in the same time period and in the close geographic area. To evaluate the PCR-RFLP assay developed here, ten strains, each isolated from four different outbreaks in different areas in Japan (Tochigi, Hyogo, Aichi, and Fukuoka prefecture), were examined to determine whether the strains in each group showed the same RFLP patterns in the PCR-RFLP assay. In accordance with the results of PFGE except for strains isolated in an area (Fukuoka), which did not produce any amplicon, ten strains in each group demonstrated the same RFLP pattern. Taken together, these data suggest that the PCR-RFLP based on region V is as useful as PFGE but perhaps more simple and rapid than PFGE for the molecular epidemiological analysis of STEC strains during sporadic and common source outbreaks.

Characterization of multidrug-resistance phenotypes and genotypes of Escherichia coli strains isolated from swine from an abattoir in Osaka, Japan

A total of 455 highly tetracycline-resistant Escherichia coli strains were isolated from 84 healthy swine from abattoirs and it was found that 56.9, 43.1, 22.2, 15.4, 2.6 and 1.5% of strains were resistant to chloramphenicol, ampicillin, kanamycin, trimethoprim-sulphamethoxazole, ofloxacin and gentamicin respectively. Interestingly, E. coli strains isolated from certain finisher hog groups exhibited resistance against 2-7 antimicrobials, but strains isolated from multiparous sow groups in each herd were resistant to only 2-4 antimicrobial agents. When randomly selected 108 tetracycline-resistant isolates were tested for the presence of resistance genes, the following genes tet(A) (n = 6), tet(B) (n = 95), tet(D) (n = 1) or both tet(A) and tet(B) (n = 6) were found to be distributed among them. Furthermore, 52 isolates carried the integrase 1 gene and 24 strains gave five different PCR amplicon profiles using primers from the variable region of integron. Extensive nucleotide sequence analyses of these amplicons revealed the presence of dhfrI, dhfrXII, dfr17, aadA, aadA2, aadA5, aadA21, aacA4 and catB3 genes which code for different antibacterial resistance proteins.

Impact of a Low-Oxygen Environment on the Efficacy of Antimicrobials against Intracellular Chlamydia trachomatis

ABSTRACT Emergence of chronic inflammation in the urogenital tract induced by Chlamydia trachomatis infection in females is a long-standing concern. To avoid the severe sequelae of C. trachomatis infection, such as pelvic inflammatory diseases (PID), ectopic pregnancies, and tubal infertility, antibiotic strategies aim to eradicate the pathogen even in asymptomatic and uncomplicated infections. Although first-line antimicrobials have proven successful for the treatment of C. trachomatis infection, treatment failures have been observed in a notable number of cases. Due to the obligate intracellular growth of C. trachomatis, reliable antimicrobial susceptibility assays have to consider environmental conditions and host cell-specific factors. Oxygen concentrations in the female urogenital tract are physiologically low and decrease further during an inflammatory process. We compared MIC testing and time-kill curves (TKC) for doxycycline, azithromycin, rifampin, and moxifloxacin under hypoxia (2% O2) and normoxia (20% O2). While low oxygen availability only moderately decreased the antichlamydial activity of azithromycin in conventional MIC testing (0.08 μg/ml versus 0.04 μg/ml; P < 0.05), TKC analyses revealed profound divergences for antibiotic efficacies between the two conditions. Thus, C. trachomatis was significantly less rapidly killed by doxycycline and azithromycin under hypoxia, whereas the efficacies of moxifloxacin and rifampin remained unaffected using concentrations at therapeutic serum levels. Chemical inhibition of multidrug resistance protein 1 (MDR-1), but not multidrug resistance-associated protein 1 (MRP-1), restored doxycycline activity against intracellular C. trachomatis under hypoxia. We suggest careful consideration of tissue-specific characteristics, including oxygen availability, when testing antimicrobial activities of antibiotics against intracellular bacteria.

The role of endoplasmic reticulum-related BiP/GRP78 in interferon gamma-induced persistent Chlamydia pneumoniae infection

Direct interaction of Chlamydiae with the endoplasmic reticulum (ER) is essential in intracellular productive infection. However, little is known about the interplay between Chlamydiae and the ER under cellular stress conditions that are observed in interferon gamma (IFN‐γ) induced chlamydial persistent infection. ER stress responses are centrally regulated by the unfolded protein response (UPR) under the control of the ER chaperone BiP/GRP78 to maintain cellular homeostasis. In this study, we could show that the ER directly contacted with productive and IFN‐γ‐induced persistent inclusions of Chlamydia pneumoniae (Cpn). BiP/GRP78 induction was observed in the early phase but not in the late phase of IFN‐γ‐induced persistent infection. Enhanced BiP/GRP78 expression in the early phase of IFN‐γ‐induced persistent Cpn infection was accompanied by phosphorylation of the eukaryotic initiation factor‐2α (eIF2α) and down‐regulation of the vesicle‐associated membrane protein‐associated protein B. Loss of BiP/GRP78 function resulted in enhanced phosphorylation of eIF2α and increased host cell apoptosis. In contrast, enhanced BiP/GRP78 expression in IFN‐γ‐induced persistent Cpn infection attenuated phosphorylation of eIF2α upon an exogenous ER stress inducer. In conclusion, ER‐related BiP/GRP78 plays a key role to restore cells from stress conditions that are observed in the early phase of IFN‐γ‐induced persistent infection.