Chlamydia trachomatis cytotoxicity associated with complete and partial cytotoxin genes.

Chlamydia trachomatis is an obligate intracellular human bacterial pathogen that infects epithelial cells of the eye and genital tract. Infection can result in trachoma, the leading cause of preventable blindness worldwide, and sexually transmitted diseases. A common feature of infection is a chronic damaging inflammatory response for which the molecular pathogenesis is not understood. It has been proposed that chlamydiae have a cytotoxic activity that contributes to this pathology, but a toxin has not been identified. The C. trachomatis genome contains genes that encode proteins with significant homology to large clostridial cytotoxins. Here we show that C. trachomatis makes a replication-independent cytotoxic activity that produces morphological and cytoskeletal changes in epithelial cells that are indistinguishable from those mediated by clostridial toxin B. A mouse chlamydial strain that encodes a full-length cytotoxin caused pronounced cytotoxicity, as did a human strain that has a shorter ORF with homology to only the enzymatically active site of clostridial toxin B. Cytotoxin gene transcripts were detected in chlamydiae-infected cells, and a protein with the expected molecular mass was present in lysates of infected epithelial cells. The protein was present transiently in infected cells during the period of cytotoxicity. Together, these data provide compelling evidence for a chlamydial cytotoxin for epithelial cells and imply that the cytotoxin is present in the elementary body and delivered to host cells very early during infection. We hypothesize that the cytotoxin is a virulence factor that contributes to the pathogenesis of C. trachomatis diseases.

The effect of oxygenated mycolic acid composition on cell wall function and macrophage growth in Mycobacterium tuberculosis.

There are three major structural classes of mycolic acids in the cell envelope of Mycobacterium tuberculosis (MTB): alpha-, methoxy- and ketomycolate. The two oxygen-containing classes are biosynthetically related through a common alpha-methyl hydroxymycolate intermediate. BCG strains that fail to produce methoxymycolate and instead produce only keto- and alpha-mycolic acids show apparent defects in the O-methyltransferase MMAS-3. Overproduction of MMAS-3 from MTB resulted in a complete replacement of ketomycolate by methoxymycolate in both BCG and MTB. In vitro growth of these recombinant strains lacking ketomycolate was impaired at reduced temperatures but appeared to be normal at 37 degrees C. Glucose uptake was significantly decreased in such strains, but uptake of chenodeoxycholate and glycine was unaffected. Although sensitivity to INH remained unchanged, these cells were found to be hypersensitive to ampicillin and rifampicin. Infectivity of BCG and H37Rv wild type or MMAS-3 overproducers in THP-1 cells was somewhat affected, but the ability of the strains lacking ketomycolate to grow within this macrophage-like cell line was severely compromised. In vivo labelling of mycolic acids during growth of H37Rv within THP-1 cells revealed a substantial increase in ketomycolate and alphamycolate synthesized by intracellularly grown mycobacteria. These results establish a critical role for mycolate composition in proper cell wall function during the growth of MTB in vivo.

The 16-kDa alpha-crystallin (Acr) protein of Mycobacterium tuberculosis is required for growth in macrophages.

Although the 16-kDa alpha-crystallin homologue of Mycobacterium tuberculosis (MTB) is the dominant protein produced by stationary phase cultures in vitro, it is undetectable in logarithmically growing cultures. By growing bacilli at defined oxygen concentrations, acr transcription was shown to be strongly induced by mildly hypoxic conditions. Acr expression also was found to be induced during the course of in vitro infection of macrophages. The acr gene was replaced with a hygromycin resistance cassette by allelic exchange in MTB H37Rv. The resulting Deltaacr::hpt strain was shown to be equivalent to wild-type H37Rv in in vitro growth rate and infectivity but was significantly impaired for growth in both mouse bone marrow derived macrophages and THP-1 cells. In addition to its proposed role in maintenance of long-term viability during latent, asymptomatic infections, these results establish a role for the Acr protein in replication during initial MTB infection.

Inhibition of a Mycobacterium tuberculosis beta-ketoacyl ACP synthase by isoniazid.

Although isoniazid (isonicotinic acid hydrazide, INH) is widely used for the treatment of tuberculosis, its molecular target has remained elusive. In response to INH treatment, saturated hexacosanoic acid (C26:0) accumulated on a 12-kilodalton acyl carrier protein (AcpM) that normally carried mycolic acid precursors as long as C50. A protein species purified from INH-treated Mycobacterium tuberculosis was shown to consist of a covalent complex of INH, AcpM, and a beta-ketoacyl acyl carrier protein synthase, KasA. Amino acid-altering mutations in the KasA protein were identified in INH-resistant patient isolates that lacked other mutations associated with resistance to this drug.

Stationary phase-associated protein expression in Mycobacterium tuberculosis: function of the mycobacterial alpha-crystallin homolog.

The majority of active tuberculosis cases arise as a result of reactivation of latent organisms which are quiescent within the host. The ability of mycobacteria to survive extended periods without active replication is a complex process whose details await elucidation. We used two-dimensional gel electrophoresis to examine both steady-state protein composition and time-dependent protein synthetic profiles in aging cultures of virulent Mycobacterium tuberculosis. At least seven proteins were maximally synthesized 1 to 2 weeks following the end of log-phase growth. One of these proteins accumulated to become a predominant stationary-phase protein. N-terminal amino acid sequencing and immunoreactivity identified this protein as the 16-kDa alpha-crystallin-like small heat shock protein. The gene for this protein was shown to be limited to the slowly growing M. tuberculosis complex of organisms as assessed by Southern blotting. Overexpression of this protein in wild-type M. tuberculosis resulted in a slower decline in viability following the end of log-phase growth. Accumulation of this protein was observed in log-phase cultures following a shift to oxygen-limiting conditions but not by other external stimuli. The protein was purified to homogeneity from overexpressing M. smegmatis in two steps and shown to have a significant ability to suppress the thermal denaturation of alcohol dehydrogenase. Collectively, these results suggest that the mycobacterial alpha-crystallin protein may play a role in enhancing long-term protein stability and therefore long-term survival of M. tuberculosis.

Ganciclovir susceptibilities of cytomegalovirus (CMV) isolates from solid organ transplant recipients with CMV viremia after antiviral prophylaxis.

Ganciclovir susceptibility studies were done on the last cytomegalovirus (CMV) isolate available from 42 solid organ transplant recipients with CMV viremia who were enrolled in a prospective CMV prophylactic trial. Viruses were categorized as follows: 13 isolates (group 1) from patients who received ganciclovir prophylaxis; 9 isolates (group 2) from patients who received acyclovir prophylaxis; 8 isolates (group 3) from patients who received acyclovir prophylaxis and ganciclovir treatment; and 12 isolates (group 4) from patients who received prophylaxis and treatment with ganciclovir. All CMV isolates were sensitive to ganciclovir (mean 50% inhibitory concentration [IC50] 1.7 microM; range, 0.2-5.3 microM). Mean IC50 values (in microM) were 1.7 for group 1 isolates, 1.2 for group 2 isolates, 2.2 for group 3 isolates, and 1.7 for group 4 isolates (P > .05). Thus, acyclovir or ganciclovir prophylaxis in solid organ transplant patients did not select ganciclovir-resistant isolates of CMV.

Acyclovir susceptibilities of herpes simplex virus strains isolated from solid organ transplant recipients after acyclovir or ganciclovir prophylaxis.

We determined the acyclovir (ACV) susceptibilities of herpes simplex virus (HSV) isolates (n = 18) recovered from solid organ transplant patients after antiviral prophylaxis with ACV or ganciclovir. All isolates tested were susceptible to ACV (50% inhibitory concentration, < 1 microM). A clinical review of patients with HSV disease showed that all improved with specific anti-HSV therapy, and no recurrences were reported.

Acyclovir treatment of varicella in otherwise healthy children.

STUDY OBJECTIVE: To determine whether acyclovir administered orally affects the duration and severity of varicella in otherwise normal children. DESIGN: Randomized, placebo-controlled, double-blind trial. SETTING: Patients' residence and university hospital clinic. PATIENTS: One hundred five children between 5 and 16 years of age with laboratory-confirmed varicella entered the study. Of the 102 who were included in the final analysis, 50 received acyclovir and 52 received placebo. INTERVENTION: Placebo or acyclovir was given orally four times daily, for 5 to 7 days. The acyclovir dose was adjusted as follows: 5 to 7 years of age, 20 mg/kg; 7 to 12 years, 15 mg/kg; and 12 to 16 years, 10 mg/kg. MEASUREMENTS AND MAIN RESULTS: Acyclovir recipients, compared with the placebo group, defervesced sooner (median, 1 day vs 2 days; p = 0.001), experienced onset of cutaneous healing sooner, as reflected by a decrease in number of lesions (median, 3 days vs 2 days; p = 0.002), and had fewer skin lesions (median, 500 vs 336; p = 0.02). Acyclovir did not significantly change the rate of complications of varicella (10% in the acyclovir group vs 13.5% among placebo subjects). Adverse drug effects were not observed. Acyclovir recipients had lower geometric mean serum antibody titers to varicella-zoster virus than their placebo counterparts 4 weeks after the onset of illness, but antibody titers in both groups were similar 1 year later. CONCLUSIONS: These results provide evidence that acyclovir is useful and well tolerated for treatment of varicella in otherwise healthy children.

Laboratory studies of acute varicella and varicella immune status.

We evaluated varicella-zoster virus (VZV) culture and serum antibody methods utilizing specimens from 620 children enrolled in protocols for prevention or treatment of varicella and samples routinely submitted to the Clinical Virology Laboratory. In a foreskin fibroblast tube culture system, we initially isolated VZV from only 29 (51%) of 57 children cultured on the first day of varicella. After modifying the method, the proportion of culture-positive children increased significantly to 36 (80%) of 45 (p less than 0.005 by corrected X2), and the median days-to-positivity were significantly shortened from 5.6 to 3.8 days (p less than 0.001, Wilcoxon rank-sum test). The Viran fluorescent antibody to membrane antigen (FAMA) assay was difficult to read and not reproducible. The standard FAMA was more sensitive than the Merck ELISA antibody test for detecting vaccine-induced antibody. The Whittaker ELISA did not detect vaccine-induced antibody but was comparable to FAMA for immune status testing (sensitivity, 95%; specificity, 92%) and for diagnosis of acute varicella.