The cell envelope architectures and cytoplasmic structures of Mycobactenium aurum CIPT 1210005, M. fortuitum, M. phlei 425, and M. thermoresistible ATCC 19527 were compared by conventional embedding and freeze-substitution methods. To ascertain the integrity of cells during each stage of the processing regimens, [1-_4Cjacetate was incorporated into the mycolic acids of mycobacterial walls, and the extraction of labeled mycolic acids was monitored by liquid scintillation counting. Radiolabeled mycolic acids were extracted by both processing methods; however, freeze-substitution resulted in the extraction of markedly less radiolabel. During conventional processing of cells, most of the radiolabel was extracted during the dehydration stage, whereas postsubstitution washes in acetone yielded the greatest loss of radiolabel during freeze-substitution. Conventional embedding frequently produced cells with condensed fibrous nucleoids and occasional mesosomes. Their cell walls were relatively thick (-25 nm) but lacked substance. Freeze-substituted cells appeared more robust, with well-dispersed nucleoids and ribosomes. The walls of all species were much thinner than those of their conventionally processed counterparts, but these stained well, which was an indication of more wall substance; the fabric of these walls, in particular the plasma membrane, appeared highly condensed and tightly apposed to the peptidoglycan. Some species possessed a thick, irregular outer layer that was readily visualized in the absence of exogenous stabilizing agents by freeze-substitution. Since freeze-substituted mycobacteria retained a greater percentage of mycolic acids in their walls, and probably other labile wall and cytoplasmic constituents, we believe that freeze-substitution provides a more accurate image of structural organization in mycobacteria than that achieved by conventional procedures.Tuberculosis remains a major health problem for humans, particularly in developing countries, with an estimated 8 million cases and 3 million deaths per year worldwide (7). In the United States, more than 22,000 new cases are being reported annually in particular populations. The epidemiology of tuberculosis has undergone a profound transition in recent years (21). An increase in the incidence of infection due to human immunodeficiency virus has resulted in a rise in the number of both typical and atypical mycobacterial infections. The Mycobacterium avium complex and M. kansasii are the most frequently encountered etiologic agents of atypical mycobacterial infections associated with AIDS (21); however, M. xenopi, M. fortuitum, and M. chelonae have also been reported previously (21).The molecular and structural compositions of the mycobacterial cell wall must play a significant role in its mechanisms of pathogenicity (18,22,23). Moreover, the intricate physical relationships among the diverse macromolecules within the wall appear to be responsible for properties such as drug resistance (4), acid fastness (1), hydrophobic interactions (25), and th...

An in vitro coculture model system was used to explore conditions that trigger neutrophil chemotaxis to Chlamydia trachomatis infected human epithelial cells (HEC-1B). Polarized HEC-1B monolayers growing on extracellular matrix (ECM) were infected with C. trachomatis serovar E. By 36 h, coincident with the secretion of chlamydial lipopolysaccharide and major outer membrane protein to the surfaces of infected cells, human polymorphonuclear neutrophils (PMNL) loaded with azithromycin migrated through the ECM and infiltrated the HEC-1B monolayer. Bioreactive azithromycin was delivered by the chemotactic PMNL to infected epithelial cells in concentrations sufficient to kill intracellular chlamydiae. However, residual chlamydial envelopes persisted for 4 weeks, and PMNL chemotaxis was triggered to epithelial cells containing residual envelopes. Infected endometrial cells demonstrated up-regulation of ENA-78 and GCP-2 chemokine mRNA. Thus, despite appropriate antimicrobial therapy, residual chlamydial envelope antigens may persist in infected tissues of culture-negative women and provide one source for sustained inflammation.

Numerous investigations have shown that 70-kDa heat shock protein (Hsp70) homologs interact tightly with hydrophobic proteins and functionally assist proteins in membranous organelles and environments. One such protein is the Chlamydia trachomatis Hsp70 that is associated with isolated outer membrane complexes of infectious elementary bodies (EB). Previous observations have indicated that chlamydial Hsp70 plays a role in EB attachment to, or entry into, endometrial epithelial cells. In this study, immunofluorescence microscopy and transmission electron microscopy observations showed that chlamydial Hsp70 is not a surface-displayed ligand on purified EB. However, brief exposure of EB to the thiol reducing agent dithiothreitol (DTT) led to surface accessibility of the Hsp70 substrate-binding domain. Reduction of the highly disulfide-cross-linked EB outer membrane proteins with DTT resulted in a decrease in EB attachment and infectivity. Interestingly, exposure of EB to the membrane-impermeable thiol-alkylating reagent 5,5-dithiobis(2-nitrobenzoic acid) enhanced attachment but compromised infectivity, suggesting that EB outer membrane proteins must be reduced for entry and productive infection. Together, our data suggest that (i) the structural integrity of the EB outer membrane, maintained by protein disulfide bonds, is important during the initial stages of attachment; (ii) reduction occurs within the localized microenvironment of host cell surfaces once intimate contact is established between EB and host cells; and (iii) subsequent conformational changes in EB ultrastructure allow productive infection in host cells. The accessibility of the Hsp70 substrate-binding domain may support the hypothesis that this protein plays a role in events following the initial stage of attachment instead of serving as a primary, surface-displayed adhesin.A distinguishing feature of the chlamydiae is their transition between infectious elementary bodies (EB) that bind to and enter host cells and noninfectious reticulate bodies that replicate intracellularly within a membrane-bound inclusion. EB are small (diameter, 300 nm) particles with an unusually rigid ultrastructure due to cysteine-rich membrane proteins that exhibit intra-and intermolecular disulfide cross-linking in the envelope (22,36). Several chlamydial molecules, including the cysteine-rich proteins, have been examined to determine their roles in EB attachment to eukaryotic cells (25,26,38,43,46,48,49,50,51). There is evidence that both the 60-kDa cysteinerich membrane protein and the major outer membrane protein (MOMP) serve as receptors for sulfated glycosaminoglycans in a tethering event between EB and host cell surfaces (46,48,49). Adherence mediated by the 40-kDa MOMP appears to be initiated by charge-charge interactions involving surface-exposed domains (50) and a high-mannose oligomannose oligosaccharide (26). It is likely that additional, unidentified surface components also participate in establishing contact between EB and the host cell surface.Although many d...

Precise localization of penicillin-binding protein (PBP)-antibiotic complexes in a methicillin-sensitive Staphylococcus aureus strain (BB255), its isogenic heterogeneous methicillin-resistant transductant (BB270), and a homogeneous methicillin-resistant strain (Col) was investigated by high-resolution electron microscopy. A mercury-penicillin V (Hg-pen V) derivative was used as a heavy metal-labeled, electron-dense probe for accurately localizing PBPs in situ in single bacterial cells during growth. The most striking feature of thin sections was the presence of an abnormally large (17 to 24 nm in width) splitting system within the thick cross walls or septa of Hg-pen V-treated bacteria of all strains. Untreated control cells possessed a thin, condensed splitting system, 7 to 9 nm in width. A thick splitting system was also distinguishable in unstained thin sections, thereby confirming that the electron contrast of this structure was not attributed to binding of bulky heavy metal stains usually used for electron microscopy. Biochemical analyses demonstrated that Hg-pen V bound to isolated plasma membranes as well as sodium dodecyl sulfate-treated cell walls and that two or more PBPs in each strain bound to this antibiotic. In contrast, the splitting system in penicillin V-treated bacteria was rarely visible after 30 min in the presence of antibiotic. These findings suggest that while most PBPs were associated with the plasma membrane, a proportion of PBPs were located within the fabric of the cell wall, in particular, in the splitting system. Inhibition of one or more high-M r PBPs by ␤-lactam antibiotics modified the splitting system and cross-wall structure, therefore supporting a role for these PBPs in the synthesis and architectural design of these structures in S. aureus.Penicillin-binding proteins (PBPs) catalyze the final stages of peptidoglycan synthesis, which is necessary for maintenance of bacterial cell morphology and growth (37). In earlier work (27), we demonstrated that PBPs of Escherichia coli could be accurately localized in situ in individual bacterial cells by using a heavy metal-labeled derivative of penicillin, mercury-penicillin V (Hg-pen V), and high-resolution electron microscopy (EM). The key to visualization was the covalent attachment of a mercury atom to the secondary ring of the ␤-lactam nucleus in order to enhance its electron density and facilitate visualization at the EM level. Since ␤-lactam antibiotics are covalently bound to PBPs, forming a stable, acylated complex, this stable complex was able to survive the often harsh fixation and embedding methods used during sample preparation, and it revealed the sites of PBP-Hg-pen V complexes as electrondense particles positioned on the plasma membranes and within the periplasm of thin-sectioned E. coli cells.We now explore the utility of Hg-pen V for the identification and localization of PBPs in a gram-positive organism. Staphylococcus aureus was selected as our gram-positive model because this organism exhibits distinct morphological ...

An in-vitro model was designed to evaluate whether polymorphonuclear leucocytes (PMN) loaded with azithromycin could migrate and deliver the antibiotic in a bioactive form to chlamydia inclusions in polarized human endometrial epithelial (HEC-1B) cells infected with Chlamydia trachomatis. PMN chemotaxis through the extracellular matrix and between infected epithelial cells was readily observed if the HEC-1B cells had been infected with chlamydiae for 36 or 48 h. Inclusions in infected epithelial cells exposed to PMN loaded with azithromycin were initially distinguished by deformed reticulate bodies and an excessive amount of chlamydial outer membrane vesicles. As the amount of PMN-delivered antibiotic increased, chlamydial inclusions were filled with large cell envelope 'ghosts' which were the remnants of lysed reticulate bodies. The lethal effect of azithromycin was confirmed by a reduction in the viability of infectious progeny. Our results demonstrate that the damage to chlamydiae was due to transport and delivery of azithromycin by PMN to infected genital epithelial cells. When infected HEC-1B cells were exposed to PMN not loaded with the antibiotic, chlamydial morphology was not obviously affected yet few viable progeny could be recovered. In this case, PMN-induced damage to host epithelial cells probably interrupted chlamydial nutrient acquisition and subsequent maturation and formation of infectious progeny.

Haemophilus ducreyi is the etiologic agent of the sexually transmitted genital ulcer disease chancroid. Predominantly a cutaneous pathogen, H. ducreyi is present in chancroid ulcers that are characterized by extensive neutrophil accumulation in intraepidermal lesions accompanied by a mononuclear infiltrate in the dermis. We used an in vitro human skin model composed of foreskin fibroblasts and keratinocytes to examine host skin cell interactions with H. ducreyi 35000. Bacteria replicated and persisted in artificial skin for at least 14 days. We observed H. ducreyi inside suprabasal keratinocytes using transmission electron microscopy. Although no bacteria were seen in the basal keratinocyte region, these cells were disrupted in infected cocultures.H. ducreyi infection stimulated increased secretion of interleukin-6 (IL-6) and IL-8 by skin cells. Conversely, tumor necrosis factor alpha and IL-1α levels were not elevated. IL-8 produced in response to H. ducreyi infection may be involved in recruiting polymorphonuclear leukocytes and other inflammatory cells, thereby contributing to the tissue necrosis and ulcer formation characteristic of chancroid.

The precise ultrastructural localization of penicillin-binding protein (PBP)-antibiotic complexes in Escherichia coli JM101, JM1O1(pBS96), and JM1O1(pPH116) was investigated by high-resolution electron microscopy. We used mercury-penicillin V (Hg-pen V) as a heavy-metal-labeled, electron-dense probe for accurately localizing PBPs in situ in single bacterial cells grown to exponential growth phase. Biochemical data derived from susceptibility tests and bacteriolysis experiments revealed no significant differences between Hg-pen V and the parent compound, penicillin V, or between strains. Both antibiotics revealed differences in the binding affinities for PBPs of all strains. Deacylation rates for PBPs were slow despite the relatively low binding affinities of antibiotics. Cells bound most of the Hg-pen V added to cultures, and the antibiotic-PBP complex could readily be seen by electron microscopy of unstained whole mounts as distinct, randomly situated electron-dense particles.Fifty to 60%6 of the antibiotic was retained by cells during processing for conventional embedding so that thin sections could also be examined. These revealed similar electron-dense particles located predominantly on the plasma membrane and less frequently in the cytoplasm. Particles positioned on the plasma membranes were occasionally shown to protrude into the periplasmic space, thereby reflecting the high resolution of the Hg-pen V probe. Moreover, some particles were observed free in the periplasm, suggesting, for the first time, that a proportion of PBPs may not be restricted to the plasma membrane but may be tightly associated with the peptidoglycan for higher efficiency of peptidoglycan assembly. All controls were devoid of the electron-dense particles. The presence of electron-dense particles in cells of the wild-type JM101, demonstrated that our probe could identify PBPs in naturally occurring strains without inducing PBP overproduction.

Safe and effective vaginal microbicidal compounds are being sought to offer women an independent method for protection against transmission of sexually acquired pathogens. The purpose of this study was to examine the efficacy of two formulations of one such compound, C31G, against Chlamydia trachomatis serovar E alone, its host epithelial cell (HEC-1B) alone, and against chlamydiae-infected HEC-1B cells. Preexposure of isolated, purified infectious chlamydial elementary bodies (EB) to C31G, at pHs 7.2 and 5.7, for 1 h at 4 degrees C resulted in reduced infectivity of EB for HEC-1B cells. Examination of the C31G-exposed 35S-EB on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographs and by Western blotting revealed a C31G concentration-dependent and pH-dependent destabilization of the chlamydial envelope, resulting in the release of chlamydial lipopolysaccharide and proteins. Interestingly, when the host human genital columnar epithelial cells were infected with chlamydiae and then exposed to dilute concentrations of C31G which did not alter epithelial cell viability, chlamydial infectivity was also markedly reduced. C31G gained access to the developing chlamydial inclusion causing damage to or destruction of metabolically active reticulate bodies as well as apparent alteration of the inclusion membrane, which resulted in premature escape of chlamydial antigen to the infected epithelial surface. These studies show that the broad-spectrum antiviral and antibacterial microbicide C31G also has antichlamydial activity.