Regulation of Mycobacterium tuberculosis whiB3 in the mouse lung and macrophages.

Mycobacterium tuberculosis is a highly successful human pathogen, with approximately 2x10(9) individuals infected globally. To understand the responses of M. tuberculosis to the in vivo environment, we studied the in vivo regulation of M. tuberculosis genes whose M. marinum homologs are induced in chronically infected frog tissues. The expression of 16S rRNA was shown to remain constant in M. tuberculosis under in vivo and in vitro conditions and therefore could be used for internal normalization in quantitative reverse transcription-PCR assays. We found whiB3, a putative transcriptional regulator implicated in mediating tissue damage, to be maximally induced at 2 weeks postinfection in the lungs of wild-type and immunodeficient (gamma interferon receptor-/-, Rag1-/-, and tumor necrosis factor alpha-/-) mice. At later time points in wild-type mice, whiB3 induction was decreased and gradually declined over the course of infection. In immunodeficient mice, whiB3 induction declined rapidly and was completely abolished in moribund animals. whiB3 was also found to be induced in naïve bone marrow-derived macrophages after 6 h of infection. whiB3 expression in vivo and in vitro was found to be inversely correlated with bacterial density. These results indicate that M. tuberculosis regulates the expression of whiB3 in response to environmental signals present in vivo and are consistent with a model of regulation by quorum sensing.

Luciferase reporter mycobacteriophages for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis in Mexico.

The utility of luciferase reporter mycobacteriophages (LRPs) for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis was prospectively evaluated in a clinical microbiology laboratory in Mexico City, Mexico. Five hundred twenty-three consecutive sputum samples submitted to the laboratory during a 5-month period were included in this study. These specimens were cultivated in Middlebrook 7H9 (MADC), MGIT, and Löwenstein-Jensen (LJ) media. Of the 71 mycobacterial isolates recovered with any of the three media, 76% were detected with the LRPs, 97% were detected with the MGIT 960 method, and 90% were detected with LJ medium. When contaminated specimens were excluded from the analysis, the LRPs detected 92% (54 of 59) of the cultures. The median time to detection of bacteria was 7 days with both the LRPs and the MGIT 960 method. LRP detection of growth in the presence of p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) was used for selective identification of M. tuberculosis complex (MTC) and compared to identification with BACTEC 460. Using the LRP NAP test, 47 (94%) out of 50 isolates were correctly identified as tuberculosis complex. The accuracy and speed of LRP antibiotic susceptibility testing with rifampin, streptomycin, isoniazid, and ethambutol were compared to those of the BACTEC 460 method, and discrepant results were checked by the conventional proportion method. In total, 50 MTC isolates were tested. The overall agreement between the LRP and BACTEC 460 results was 98.5%. The median LRP-based susceptibility turnaround time was 2 days (range, 2 to 4 days) compared to 10.5 days (range, 7 to 16 days) by the BACTEC 460 method. Phage resistance was not detected in any of the 243 MTC isolates tested. Mycobacteriophage-based approaches to tuberculosis diagnostics can be implemented in clinical laboratories with sensitivity, specificity, and rapidity that compare favorably with those of the MGIT 960 and BACTEC 460 methods. The phages currently provide the fastest phenotypic assay for susceptibility testing.

Rat neutrophil defensins. Precursor structures and expression during neutrophilic myelopoiesis.

Defensins constitute a family of 3- to 4-kDa antimicrobial peptides that are stored in the cytoplasmic granules of neutrophils, some macrophages, and intestinal Paneth cells. We have assessed defensin gene expression during myeloid differentiation by first characterizing cDNAs for each of the four known rat neutrophil defensins (RatNP 1-4). The cDNA sequences revealed that the peptides are synthesized as 87-94 amino acid precursors, each containing signal, pro-, and mature peptide segments. RatNP-3 and -4 mRNAs, but not those for RatNP-1 and -2 or other myeloid defensins, contained unique polypurine tracts located close to the termination codon in the 3' untranslated region. By using cDNA probes and/or riboprobes, we evaluated defensin transcript levels in a variety of tissues and in the full spectrum of neutrophil precursors. By in situ hybridization, defensin mRNAs were localized to neutrophil precursors in the bone marrow, with the highest mRNA levels occurring in promyelocytes and somewhat lower signals occurring in myeloblasts and myelocytes. Defensin mRNAs were not detectable in bands or mature neutrophils, nor at significant levels in tissues other than bone marrow. The accumulation of defensin protein in bone marrow cells was assessed by immunohistochemical staining with anti-RatNP-1 Ab. RatNP 1-4 mRNAs and protein levels were then correlated for each stage of neutrophilic differentiation to reveal the maturational profile of myeloid defensin gene expression in the rat.