Soraya L. Moghazeh

Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule.

The production of most toxins and other exoproteins in Staphylococcus aureus is controlled globally by a complex polycistronic regulatory locus, agr. Secretory proteins are up‐regulated by agr whereas surface proteins are down‐regulated. agr contains two divergent promoters, one of which directs the synthesis of a 514 nucleotide (nt) transcript, RNAIII. In this report, we show that the cloned RNAIII determinant restores both positive and negative regulatory functions of agr to an agr‐null strain and that the RNA itself, rather than any protein, is the effector molecule. RNAIII acts primarily on the initiation of transcription and, secondarily in some cases, at the level of translation. In these cases, translation and transcription are regulated independently. RNAIII probably regulates translation directly by interacting with target gene transcripts and transcription indirectly by means of intermediary protein factors.

Theagr P2 operon: An autocatalytic sensory transduction system inStaphylococcus aureus

The synthesis of virulence factors and other exoproteins inStaphylococcus aureus is controlled by the global regulator,agr. Expression of secreted proteins is up-regulated in the postexponential growth phase, whereas expression of surface proteins is down-regulated byagr. Theagr locus consists of two divergent operons, transcribed from neighboring but non-overlapping promoters, P2 and P3. The P2 operon sequence, reported here, contains 4 open reading frames,agr A, C, D, andB, of whichA andC appear to encode proteins of a classical 2-component signal transduction pathway. The P3 operon specifies a 0.5 kb transcript, RNA III, which is the actual effector of theagr response, and, incidentally, encodes theagr-regulated peptide δ-hemolysin. Transcriptional fusions have shown that both P2 and P3 areagr sensitive (function in anagr+ but not in anagr background) and deletion analysis has shown that all four of the P2 ORFs are involved;agrA andagrC seem to be absolutely required for the transcriptional activation of theagr locus, whereasagrB andagrD seem to be partially required. Since transcription of P2 requires P2 operon products, the P2 operon is autocatalytic, and is thus admirably suited to the need for rapid production of exo-proteins at a time when overall growth is coming to a halt.

Molecular Genetic Analysis of Nucleotide Polymorphisms Associated with Ethambutol Resistance in Human Isolates of Mycobacterium tuberculosis

ABSTRACT Ethambutol (EMB) is a central component of drug regimens used worldwide for the treatment of tuberculosis. To gain insight into the molecular genetic basis of EMB resistance, approximately 2 Mb of five chromosomal regions with 12 genes in 75 epidemiologically unassociated EMB-resistant and 33 EMB-susceptible Mycobacterium tuberculosis strains isolated from human patients were sequenced. Seventy-six percent of EMB-resistant organisms had an amino acid replacement or other molecular change not found in EMB-susceptible strains. Thirty-eight (51%) EMB-resistant isolates had a resistance-associated mutation in only 1 of the 12 genes sequenced. Nineteen EMB-resistant isolates had resistance-associated nucleotide changes that conferred amino acid replacements or upstream potential regulatory region mutations in two or more genes. Most isolates (68%) with resistance-associated mutations in a single gene had nucleotide changes in embB, a gene encoding an arabinosyltransferase involved in cell wall biosynthesis. The majority of these mutations resulted in amino acid replacements at position 306 or 406 of EmbB. Resistance-associated mutations were also identified in several genes recently shown to be upregulated in response to exposure of M. tuberculosis to EMB in vitro, including genes in theiniA operon. Approximately one-fourth of the organisms studied lacked mutations inferred to participate in EMB resistance, a result indicating that one or more genes that mediate resistance to this drug remain to be discovered. Taken together, the results indicate that there are multiple molecular pathways to the EMB resistance phenotype.

Comparative sequence and functional analysis of pT181 and pC221, cognate plasmid replicons from Staphylococcus aureus

SummaryThe nucleotide sequence of pC221, a 4.6 kb Staphylococcus aureus plasmid is presented. The replication region of the plsmid is identified and compared with the corresponding region of pT181, a compatible but related plasmid. Both plasmids encode trans-active replicon-specific initiator proteins, RepC for pT181 and RepD for pC221. Plasmid replication rate is controlled by regulation of the rate of synthesis of the inititator protein by means of inhibitory 5′ countertranscripts. Key elements of the control system are closely conserved between the two plasmids whereas less critical elements show extensive divergence. Overall architecture is also conserved, suggesting functional parallelism.The replication origin for both plasmids is contained within the N-terminal region of the initiator protein coding sequence; the two coding sequences are highly homologous but have two important areas of divergence, one within the origin region, the other near the C-terminus. In vivo recombinants between the two plasmids isolated previously (Iordanescu 1979) have crossover points within the initiator gene, between the two divergent regions. The recombinant plasmids have hybrid initiator proteins and are defective for replication, requiring the simulaneous presence of the parental plasmid from which their origin is derived. They are able to complement replication-defective mutants of the other parental plasmid, suggesting that the recognition specificity of the hybrid initiator protein resides in its C-terminal end and that the specific recognition site for the protein corresponds to the divergent region within the origin.

Inactivation of mprF affects vancomycin susceptibility in Staphylococcus aureus.

A chemically generated mutant of Staphylococcus aureus RN4220, GC6668, was isolated that had a fourfold increase in resistance to vancomycin. This phenotype reverted back to susceptibility by insertional mutagenesis with Tn917. In a selected set of revertants, Tn917 insertion was mapped to a unique chromosomal region upstream of mprF, a recently described gene that determines staphylococcal resistance to several host defense peptides. The genetic linkage between the vancomycin susceptibility and Tn917 insertion was then confirmed by transduction backcrosses into both GC6668 and GISA isolates, MER-S12 and HT2002 0127. Northern blot analysis, insertional inactivation and complementation experiments showed that mprF mediates vancomycin susceptibility in S. aureus. The inactivation of mprF by Tn917 insertion in HT2002 0127 caused a significant increase in the binding of vancomycin to the cell membranes. This observation serves as a likely mechanism of the increased vancomycin susceptibility associated with mprF inactivation.

A rapid method to quantitate non-labeled RNA species in bacterial cells

We have developed a rapid method to quantitate specific bacterial RNA species. The method measures the steady-state level of RNA, produces a linear response over more than a 16-fold range of RNA concentration, and can be used for Staphylococcus aureus, Escherichia coli and Bacillus subtilis. In this method, a sheared whole-cell lysate of approx. 7 x 10(8) organisms, prepared as for plasmid screening, is separated on agarose, blotted to a nitrocellulose filter, hybridized with a radiolabeled DNA probe, and autoradiographed. The RNA species are quantitated by counting the radioactive bands on the filter. We have applied the method to the measurement of mRNA induction of the genes encoding beta-lactamase, ermC rRNA methylase, and the alpha-complementing fragment of beta-galactosidase. Upon induction, a ten-fold increase in the mRNA for each gene was observed. The peak mRNA level occurred after 30 min for beta-lactamase, 20 min for beta-galactosidase, and 5 min for the ermC rRNA methylase.

Identification and Evolution of an IS6110 Low-Copy-Number Mycobacterium tuberculosis Cluster

A cohort of 56 patients infected with related strains of Mycobacterium tuberculosis, the S75 group, was identified in a New Jersey population-based study of all isolates with a low number of copies of the insertion element IS6110. Genotyping was combined with surveillance data to identify the S75 group and to elucidate its recent evolution. The S75 group had similar demographic and geographic characteristics. Seventeen persons (30%) were linked epidemiologically. The S75 group was segregated from other low-copy-number isolates on the basis of several independent molecular methods. This group included 3 IS6110 genotype variants: BE, H6, and C28, containing 1, 2, and 3 IS6110 insertions, respectively. IS6110 insertion site mapping and comparative sequence analysis strongly suggest a stepwise acquisition of IS6110 elements from BE to H6 to C28. S75 represents a locally produced strain cluster that has recently evolved. The combination of multiple molecular tools with traditional epidemiology provides novel insights into dissemination, local transmission, and evolution of M. tuberculosis.

The Evolution of Drug Resistance in Mycobacterium tuberculosis: From a Mono–Rifampin-Resistant Cluster into Increasingly Multidrug-Resistant Variants in an HIV-Seropositive Population

We describe the genotypic and phenotypic characteristics of a mono-rifampin-resistant (RIF(R)) Mycobacterium tuberculosis strain cluster (designated AU-RIF(R)) and the acquisition of additional drug resistance. Drug susceptibility, sequences of regions that determine drug resistance, and basic clinical data were examined. A rare codon duplication (514(TTC)) in rpoB conferring high levels of RIF(R) (minimum inhibitory concentration of >256 microg/mL) in 29 isolates was identified. AU-RIF(R) strains developed secondary resistance to isoniazid and 7 resistance combinations to 6 different antibiotics. Patients infected with AU-RIF(R) strains were primarily immunocompromised. These data suggest that host factors, such as HIV status, may allow dissemination of mono-RIF(R) strains and facilitate the accumulation of additional drug resistance.

Clinical and Laboratory Findings of Tuberculous Meningitis (TBM) Patients(pts) in Cairo, Egypt |[diams]| 818

We retrospectively studied the clinical and laboratory findings of 23 culture positive TBM pts by comparing their clinical characteristics with their isolates IS6110-based DNA fingerprints (van Embden, 1993) and drug susceptibility.