Identification of a Membrane Protein Required for Lipomannan Maturation and Lipoarabinomannan Synthesis in Corynebacterineae.

Mycobacterium tuberculosis and related Corynebacterineae synthesize a family of lipomannans (LM) and lipoarabinomannans (LAM) that are abundant components of the multilaminate cell wall and essential virulence factors in pathogenic species. Here we describe a new membrane protein, highly conserved in all Corynebacterineae, that is required for synthesis of full-length LM and LAM. Deletion of the Corynebacterium glutamicum NCgl2760 gene resulted in a complete loss of mature LM/LAM and the appearance of a truncated LM (t-LM). Complementation of the mutant with the NCgl2760 gene fully restored LM/LAM synthesis. Structural studies, including monosaccharide analysis, methylation linkage analysis, and mass spectrometry of native LM species, indicated that the ΔNCgl2760 t-LM comprised a series of short LM species (8-27 residues long) containing an α1-6-linked mannose backbone with greatly reduced α1-2-mannose side chains and no arabinose caps. The structure of the ΔNCgl2760 t-LM was similar to that of the t-LM produced by a C. glutamicum mutant lacking the mptA gene, encoding a membrane α1-6-mannosyltransferase involved in extending the α1-6-mannan backbone of LM intermediates. Interestingly, NCgl2760 lacks any motifs or homology to other proteins of known function. Attempts to delete the NCgl2760 orthologue in Mycobacterium smegmatis were unsuccessful, consistent with previous studies indicating that the M. tuberculosis orthologue, Rv0227c, is an essential gene. Together, these data suggest that NCgl2760/Rv0227c plays a critical role in the elongation of the mannan backbone of mycobacterial and corynebacterial LM, further highlighting the complexity of lipoglycan pathways of Corynebacterineae.

Transcriptional profiling of innate and adaptive human immune responses to mycobacteria in the tuberculin skin test.

The tuberculin skin test (TST) is a model of integrated innate and adaptive human immune responses to Mycobacterium tuberculosis, but the component processes that are involved in this model have not previously been defined in vivo. We used transcriptional profiling to study these responses within the TST at molecular and system levels. Skin biopsies from TST injection sites were examined in subjects classified as TST(+) or TST(-) by clinical and histological criteria. Genome-wide expression arrays showed evolution of immune responses reflecting T-cell activation and recruitment with uniquely Th1-polarized responses and cytotoxic T cells (CTLs). In addition, distinct innate immune and IFN-γ-stimulated gene expression signatures were identified, under the regulation of NF-κB and STAT1 transcriptional control. These were highly enriched for chemokines and MHC class II molecules providing a potential mechanism for paracrine amplification of inflammatory responses in the TST, by supporting cellular recruitment and enhancing antigen presentation. The same repertoire of innate and adaptive immune responses was evident in TST(+) and TST(-) subjects alike, clinically positive TSTs being distinguished only by quantitatively greater differences. These data provide new insights into complex multifaceted responses within the TST, with much greater sensitivity than previous clinical or histological assessments.

MmpA, a Conserved Membrane Protein Required for Efficient Surface Transport of Trehalose Lipids in Corynebacterineae.

Cell walls of bacteria of the genera Mycobacterium and Corynebacterium contain high levels of (coryno)mycolic acids. These very long chain fatty acids are synthesized on the cytoplasmic leaflet of the inner membrane (IM) prior to conjugation to the disaccharide, trehalose, and transport to the periplasm. Recent studies on Corynebacterium glutamicum have shown that acetylation of trehalose monohydroxycorynomycolate (hTMCM) promotes its transport across the inner membrane. Acetylation is mediated by the membrane acetyltransferase, TmaT, and is dependent on the presence of a putative methyltransferase, MtrP. Here, we identify a third protein that is required for the acetylation and membrane transport of hTMCM. Deletion of the C. glutamicum gene NCgl2761 (Rv0226c in Mycobacterium tuberculosis) abolished synthesis of acetylated hTMCM (AcTMCM), resulting in an accumulation of hTMCM in the inner membrane and reduced synthesis of trehalose dihydroxycorynomycolate (h2TDCM), a major outer membrane glycolipid. Complementation with the NCgl2761 gene, designated here as mmpA, restored the hTMCM:h2TDCM ratio. Comprehensive lipidomic analysis of the ΔtmaT, ΔmtrP and ΔmmpA mutants revealed strikingly similar global changes in overall membrane lipid composition. Our findings suggest that the acetylation and membrane transport of hTMCM is regulated by multiple proteins: MmpA, MtrP and TmaT, and that defects in this process lead to global, potentially compensatory changes in the composition of inner and outer membranes.

Epidemiology of HIV-1 subtypes among men who have sex with men in Cape Town, South Africa.

OBJECTIVE: Early studies in Cape Town identified independent HIV-1 epidemics, with distinct viral subtypes, among men who have sex with men (MSM) and the heterosexual population. However, few recent HIV-1 subtype data are available for MSM in South Africa. We examined HIV-1 subtypes among MSM in Cape Town. DESIGN: Cross-sectional survey. METHODS: Self-identified MSM were recruited from geographically and racially disparate communities across Cape Town. Participants completed behavioral questionnaires and underwent HIV testing. Virus isolated from infected participants underwent complete env gp160 sequencing, and HIV-1 subtypes were assigned through phylogenetic analysis. RESULTS: In total, 194 HIV-infected MSM were enrolled: 67% black African, 24% colored, and 9% white men. More black African men identified as bisexual or heterosexual compared with other races. Overall, 31%-66% of men reported a recent partner of another race. HIV-1 subtypes were confirmed for 143 participants: 81% were subtype C, 14% B, 1% A1, 1% F2, and 3 recombinant viruses. Subtype C virus was associated with black African race (P = 0.003 compared with colored; P < 0.001 compared with white), men who identified as bisexual/heterosexual (P = 0.01), and reported a female sexual partner in the last year (P = 0.02). Compared with previous studies, an increasing prevalence of subtype C virus was noted among white MSM. CONCLUSIONS: This molecular epidemiology study provides novel evidence of sexual network links between the heterosexual and MSM epidemics and between historically racially disparate communities. These findings provide insights into the drivers of HIV epidemics in different population groups and may have implications for prevention strategies.

Feasibility and diagnostic utility of antigen-specific interferon-gamma responses for rapid immunodiagnosis of tuberculosis using induced sputum.

BACKGROUND: The diagnosis of smear-negative or sputum-scarce tuberculosis (TB) is problematic as culture takes several weeks and representative biological samples are difficult to obtain. RD-1 antigen-specific interferon-gamma release assays (IGRAs) are sensitive and specific blood-based tests for the diagnosis of M. tuberculosis infection. The feasibility and diagnostic utility of this rapid immunodiagnostic assay, using cells from induced sputum, is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Cells isolated from induced sputum were co-cultured with ESAT-6 and CFP-10 antigens using a standardized enzyme-linked immunospot (ELISPOT) assay (T-SPOT.TB) in 101 consecutively recruited TB suspects or non-TB controls. An optimization phase using 28 samples was followed by a validation phase using samples from 73 participants (20 with definite or probable TB, and 48 with non-TB). Despite optimization of sputum processing 65/73 (89%) of the IGRAs in the validation phase were inconclusive. 44/73 (60%) tests failed due to sputum induction-related factors [sputum induction-related adverse events (n = 5), inadequate sputum volume (n = 8), non-homogenisable sputum (n = 7), and insufficient numbers of cells to perform the assay (n = 24)], whilst 20/73 (27%) tests failed due T-SPOT.TB assay-related factors [excessive debris precluding reading of spots in the ELISPOT well (n = 6), failure of the positive control (n = 11), or high spot count in the negative control (n = 3)]. Only 8/73 (11%) of the available samples could therefore be correctly categorized (7 definite or probable TB, and 1 non-TB patient). Thus, 13/20 (65%) of the definite or probable TB cases remained undiagnosed. CONCLUSIONS/SIGNIFICANCE: Rapid immunodiagnosis of pulmonary TB by antigen-specific IFN-gamma ELISPOT responses, using cells from induced sputum, is possible. However, the test, in its current ELISPOT format, is not clinically useful because the majority of the assays are inconclusive.