The antibiotic fosfomycin inhibits bacterial cell wall biosynthesis by inactivation of UDP-GlcNAc enolpyruvyl tranferase (MurA). Prior work has established that Cys115 of Escherichia coli and Enterobacter cloacae MurA is the active site nucleophile alkylated by fosfomycin and implicated this residue in the formation of a covalent phospholactyl-enzyme adduct derived from substrate, phosphoenolpyruvate (PEP). On the basis of sequencing information from putative MurA homolog from Mycobacterium tuberculosis, we generated a C115D mutant of E. coli MurA that was highly active but fully resistant to time-dependent inhibition by fosfomycin. Fosfomycin still bound to the active site of C115D MurA, as established by the observed reversible competitive inhibition by fosfomycin. Fosfomycin still bound to the active site of C115D MurA, as established by the observed reversible competitive inhibition vs PEP. In contrast to the broad pH-independent behavior of wild-type (WT) MurA, C115D mutant activity titrated across the pH range examined (pH 5.5-9) with an apparent pKa approximately 6, with kcatC115D ranging from approximately 10kcatWT at pH 5.5 to <0.1kcatWT at pH9.0. Km(PEP)115D was relatively constant in the pH range examined and increased approximately 100-fold relative to Km(PEP)WT. A fosfomycin-resistant C115E mutant with -1% activity of the C115D mutant was found to follow a pH dependence similar to that observed for C115D MurA. The contrasting pH dependences of WT and C115D MurA was also observed in the reaction with the pseudosubstrate, (Z)-3-fluorophosphoenolpyruvate, strongly suggesting a role for Cys/Asp115 as the general acid in the protonation of C-3 of PEP during MurA-catalyzed enol ether transfer. The difference in nucleophilicity between the carboxylate side chains of Asp115 and Glu115 and the thiolate group of Cys115 suggests that covalent enzyme adduct formation is not required for catalytic turnover and, furthermore, provides a chemical rationale for the resistance of the C115D and C115E mutants to fosfomycin inactivation.

Mycobacterium tuberculosis has innate resistance to a range of broadspectrum antimicrobial agents. This may in part reflect the relative impermeability of the mycobacterial cell wall, but additional specific mechanisms may also be important. In the case of fosfomycin, it has been suggested that a key difference in the active site of the M. tuberculosis MurA enzyme might confer resistance. In Escherichia coli, fosfomycin covalently binds to a cysteine normally involved in the enzymic activity, while protein alignments predict an aspartate at this position in the M. tuberculosis MurA. In the present study, it is demonstrated that the wild-type M. tuberculosis MurA is indeed resistant to fosfomycin, and that it becomes sensitive following replacement of the aspartate residue in position 117 by a cysteine. In addition, the study illustrates the use of an inducible expression system in mycobacteria to allow functional characterization of an M. tuberculosis enzyme that is unstable during constitutive expression.

Onset of rheumatoid arthritis (RA) is widely believed to be preceded by exposure to some environmental trigger such as bacterial infectious agents. The influence of bacteria on RA disease onset or pathology has to date been controversial, due to inconsistencies between groups in the report of bacterial species isolated from RA disease tissue. Using a modified technique of reverse transcriptase-PCR amplification, we have detected bacterial rRNA in the synovial tissue of late-stage RA and non-RA arthritis controls. This may be suggestive of the presence of live bacteria. Sequencing of cloned complementary rDNA (crDNA) products revealed a number of bacterial sequences in joint tissue from each patient, and from these analyses a comprehensive profile of the organisms present was compiled. This revealed a number of different organisms in each patient, some of which are common to both RA and non-RA controls and are probably opportunistic colonizers of previously diseased tissue and others which are unique species. These latter organisms may be candidates for a specific role in disease pathology and require further investigation to exclude them as causative agents in the complex bacterial millieu. In addition, many of the detected bacterial species have not been identified previously from synovial tissue or fluid from arthritis patients. These may not be easily cultivable, since they were not revealed in previous studies using conventional in vitro bacterial culture methods. In situ hybridization analyses have revealed the joint-associated bacterial rRNA to be both intra- and extracellular. The role of viable bacteria or their nucleic acids as triggers in disease onset or pathology in either RA or non-RA arthritis controls is unclear and requires further investigation.

Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 ‘alveolar’ cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham’s F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line.

Allelic replacement will be a vital tool for understanding gene function in mycobacteria. Disruption of the chromosomal hisD gene of Mycobacterium smegmatis by standard gene replacement methods was surprisingly difficult, with most products being caused by illegitimate recombination (IR) events. A recombination assay was therefore developed and used to optimize conditions for homologous recombination (HR) in M. smegmatis. Treatment of competent cells with UV, hydrogen peroxide or mitomycin C did not improve the frequency of HR; however, treatment of the DNA with alkali or UV enhanced recombination frequency, while boiling did not. Applying these observations to allele replacement, UV and alkali treatment of transforming DNA increased HR events with pyrF and hisD, while the level of IR was unchanged. The introduction of ss phagemid DNA improved the level of HR and abolished IR. In Mycobacterium intracellulare the use of al kali-denatured DNA increased the numbers of recombinants obtained with an inactivated 19Ag gene, while in Mycobacterium tuberculosis, inactivation of a putative haemolysin gene, tlyA, was achieved using both UV-irradiated DNA and ss phagemid DNA.Significantly, IR, which has been reported to be a problem in this species, was not observed. Thus, four genes in three species were successfully knocked-out using non-replicating DNA pretreated with alkali, UV or in an ss form. The use of these methods to enhance HR will greatly facilitate experiments to inactivate other genes in these important species.