Claudia J. Lammel

Comparative genomes of Chlamydia pneumoniae and C. trachomatis

Chlamydia are obligate intracellular eubacteria that are phylogenetically separated from other bacterial divisions. C. trachomatis and C. pneumoniae are both pathogens of humans but differ in their tissue tropism and spectrum of diseases. C. pneumoniae is a newly recognized species of Chlamydia that is a natural pathogen of humans, and causes pneumonia and bronchitis. In the United States, approximately 10% of pneumonia cases and 5% of bronchitis cases are attributed to C. pneumoniae infection. Chronic disease may result following respiratory-acquired infection, such as reactive airway disease, adult-onset asthma and potentially lung cancer. In addition, C. pneumoniae infection has been associated with atherosclerosis. C. trachomatis infection causes trachoma, an ocular infection that leads to blindness, and sexually transmitted diseases such as pelvic inflammatory disease, chronic pelvic pain, ectopic pregnancy and epididymitis. Although relatively little is known about C. trachomatis biology, even less is known concerning C. pneumoniae. Comparison of the C. pneumoniae genome with the C. trachomatis genome will provide an understanding of the common biological processes required for infection and survival in mammalian cells. Genomic differences are implicated in the unique properties that differentiate the two species in disease spectrum. Analysis of the 1,230,230-nt C. pneumoniae genome revealed 214 protein-coding sequences not found in C. trachomatis, most without homologues to other known sequences. Prominent comparative findings include expansion of a novel family of 21 sequence-variant outer-membrane proteins, conservation of a type-III secretion virulence system, three serine/threonine protein kinases and a pair of parologous phospholipase-D-like proteins, additional purine and biotin biosynthetic capability, a homologue for aromatic amino acid (tryptophan) hydroxylase and the loss of tryptophan biosynthesis genes.

Chlamydia outer membrane protein discovery using genomics

Outer membrane proteins of microbial pathogens serve essential roles in engaging the host environment and can be important immunotherapeutic targets. Because of the difficulty of growing large quantities of chlamydiae suitable for biochemical fractionation, little was known about their outer membrane protein composition prior to the recent sequencing of the C. trachomatis and C. pneumoniae genomes. Using bioinformatic approaches to characterize chlamydial open reading frames, novel outer membrane proteins were predicted. Several of the predicted outer membrane proteins recently have been shown to be translated and localized to the surface of the chlamydial outer membrane.

Immunogenicity and evolutionary variability of epitopes within IgA1 protease from serogroup A Neisseria meningitidis

Five murine epitopes were defined and mapped within IgA1 protease produced by Neisseria meningitidis. Epitopes 1 and 2 were present in IgA1 protease from all strains, and from Neisseria gonorrhoeae. Epitopes 3 through to 5 varied between subgroups of serogroup A meningococci. but have remained constant over decades within the subgroups, except for epitope 4, which changed between 1983 and 1987 during the spread of subgroup III meningococci from Asia to Africa. Binding of monoclonal antibodies to epitopes 1, 4 and 5 neutralized enzymatic function. Human sera containing antibodies to lgA1 protease as a result of natural infection inhibited binding of monoclonal antibodies to epitope 4 but not to the other epitopes.

Opa (protein II) influences gonococcal organization in colonies, surface appearance, size and attachment to human fallopian tube tissues

Opa-expressing variants of Neisseria gonorrhoeae strain F62-SF and an Opa- variant, all non-piliated, were examined for differences in the interaction of the bacteria within colonies and in attachment to and damage of human fallopian tube mucosa. Expression of certain Opas was associated with the formation of transparent colonies where the bacteria were tightly packed and evenly spaced within the colonies. Expression of other Opas was associated with the formation of opaque colonies where the gonococci were less tightly packed and were unevenly spaced. Distinct differences in the size of the gonococci and in their surface characteristics were dependent upon the Opa being expressed. Certain Opas were associated with gonococci that had significantly larger cross-sectional areas and bigger perimeters. Scanning electron microscopy showed that OpaC- and OpaD-containing variants yielded greater mucosal damage than OpaB-containing and Opa- variants with the least damage caused by the OpaA-containing variant (clumped bacteria from dark opaque friable colonies). The mucosal damage after 60 min incubation included shortening and decreased numbers of microvilli on non-ciliated cells and invagination and sloughing of ciliated cells. Differences in the interactions of gonococci within colonies and in attachment to fallopian tube mucosa and damage to the mucosal cells occurred with different Opa-expressing variants of N. gonorrhoeae strain F62-SF.

Chlamydial Genovar Distribution after Communitywide Antibiotic Treatment

Major outer membrane protein sequences, determined from Chlamydia-positive eye swab samples collected in 2 Egyptian villages, were used to analyze the epidemiology of trachoma in an endemic setting. Samples were collected during the 1999 Azithromycin in Control of Trachoma trial, in which residents of villages were mass treated with either oral azithromycin or topical tetracycline and were followed up for nearly 2 years. Three genovar families (A, Ba, and C) and 12 genovars were detected, with 2 genovars (A1 and Ba1) comprising almost 75% of the samples. The presence of >1 genovar within households was common, with > or =24% of households having >1 genovar. Evidence consistent with reinfection and persistence as mechanisms of communitywide continued presence of trachoma was provided by data for individuals infected with rare genovars.

Prevalence of gene sequences coding for hypervariable regions of Opa (protein II) in Neisseria gonorrhoeae

Opas (protein IIs) are a family of surface‐exposed proteins of Neisseria gonorrhoeae. Each strain of N. gonorrhoeae has multiple (10–11) genes encoding for Opas. Identifiable elements in opa genes include the coding repeat within the signal sequence, conserved 5′ and 3′ regions, and hypervariable regions (HV1 and HV2) located within the structural gene. N. gonorrhoeae strains appear to have many biological properties in common that are either HV‐region‐mediated or associated with the presence of specific HV regions, suggesting that HV regions could be found in many clinical isolates. Oligonucleotides from three source strains representing three conserved regions of opa, 12 HV1 regions, and 14 HV2 regions were used by dot blot analysis to probe 120 clinicial isolates of N. gonorrhoeae. The probe for the coding repeat hybridized to all 120 strains, the 3′ conserved‐region probe reacted with 98% of the strains, and the 5′ conserved‐region probe with 90% of the strains. Nine HV1 probes hybridized to 3.3–39.2% of the strains, and 13 of the HV2 probes hybridized to 1.7–25% of the isolates. Analysis of the number of probes that hybridized to each of the isolates showed that 19% did not hybridize with any of the HV1 probes and 25% did not hybridize with any of the HV2 probes. Approximately three‐quarters of the isolates hybridized with one, two or three of the HV1 probes or one, two or three of the HV2 probes; 89% of the isolates hybridized to least one HV1 or oe HV2 probe. The data indicate that some genes encoding HV regions of N. gonorhoeae Opa proteins are widely distributed in nature.

Gonococcal attachment to eukaryotic cells.

The attachment of Neisseria gonorrhoeae to eukaryotic cells grown in tissue culture was analyzed by use of light and electron microscopy and by labeling of the bacteria with [3H]- and [14C]adenine. Isogenic piliated and nonpiliated N. gonorrhoeae from opaque and transparent colonies were studied. The results of light microscopy studies showed that the gonococci attached to cells of human origin, including Flow 2000, Hela 229, and HEp 2. Studies using radiolabeled gonococci gave comparable results. Piliated N. gonorrhoeae usually attached in larger numbers than nonpiliated organisms, and those from opaque colonies attached more often than isogenic variants from transparent colonies. Day-to-day variation in rate of attachment was observed. Scanning electron microscopy studies showed the gonococcal attachment to be specific for microvilli of the host cells. It is concluded that more N. gonorrhoeae from opaque colonies, as compared with isogenic variants from transparent colonies, attach to eukaryotic cells grown in tissue culture.

Cervical secretory immunoglobulin A in adolescent girls

PURPOSE To determine whether there are differences in levels of cervical secretory immunoglobulin A (sIgA) between adolescent girls in the secretory and proliferative phases of their menstrual cycle. METHODS Sexually active adolescent girls (n = 117) at health maintenance organization (HMO) based adolescent medical clinic were recruited into the study. In addition to demographic and clinical data, cervical specimens were collected for sIgA measurement and gonorrhea culture, urine for chlamydia ligase chain reaction, and blood for progesterone levels. Subjects were classified as being in the proliferative phase or secretory phase of the menstrual cycle on the basis of their progesterone levels. RESULTS The mean age of the subjects was 17.2 years old. There was no difference in the sIgA levels between those in the proliferative phase of their cycle (n = 45; mean sIgA level, 0.0055 mg/mL) and those in the secretory phase (n = 40; mean sIgA level, 0.0032 mg/mL) (p > .10). CONCLUSIONS The secretory phase of the menstrual cycle does not appear to be associated with higher levels of sIgA in adolescent girls. These results suggest that adolescents with anovulatory cycles, i.e., those who lack a secretory phase, may not be at increased risk for genital tract infections such as chlamydia or gonorrhea.

Bactericidal action of nafcillin, vancomycin, and three cephalosporins against nafcillin-susceptible and nafcillin-resistant coagulase-negative staphylococci

Coagulase-negative staphylococci (S. epidermidis, 43 strains; S. warneri, 16 strains; S. haemolyticus, five strains; and others, four strains) were tested by the agar dilution method for nafcillin susceptibility: 53 were susceptible with a minimal inhibitory concentration (MIC) of less than or equal to 2 micrograms/ml; four were of indeterminate susceptibility, MIC = 4-16 micrograms/ml; and 11 were resistant, MIC greater than or equal to 32 micrograms/ml. The bactericidal activities from 0 to 24 hr for nafcillin, vancomycin, cephalothin, cefazolin, and cefamandole, each at 16 micrograms/ml in broth, were determined for all the isolates. The data indicate that a nafcillin agar dilution susceptibility test result of resistance does not consistently predict lack of killing activity by the cephalosporins. It is likely that each cephalosporin would have to be tested against individual coagulase-negative staphylococci in order to determine a suitable therapeutic or prophylactic cephalosporin, if a cephalosporin were to be used. Vancomycin was bactericidal for all the nafcillin-resistant coagulase-negative organisms tested.