c Phthiocerol dimycocerosates (PDIMs) and structurally related phenolic glycolipids (PGLs) are complex cell wall lipids unique to pathogenic mycobacteria. While these lipids have been extensively studied in recent years, there are conflicting reports on some aspects of their biosynthesis and on the role of PDIMs and especially PGLs in virulence of Mycobacterium tuberculosis. This has been complicated by the natural deficiency of PGLs in many clinical strains of M. tuberculosis and the frequent loss of PDIMs in laboratory M. tuberculosis strains. In this study, we isolated seven mutants of Mycobacterium marinum deficient in PDIMs and/or PGLs in which multiple genes of the PDIM/PGL biosynthetic locus were disrupted by transposon insertion. Zebrafish infection experiments showed that M. marinum strains lacking one or both of these lipids were avirulent, suggesting that both PDIMs and PGLs are required for virulence. We also found that these strains were hypersensitive to antibiotics and exhibited increased cell wall permeability. Our studies provide new insights into the biosynthesis of PDIMs/PGLs and may help us to understand the role of PDIMs and PGLs in M. tuberculosis virulence. P athogenic mycobacteria produce two structurally related, methyl-branched fatty acid-containing lipids called phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs). PDIMs and PGLs have long-chain fatty acid backbones consisting of 3-methoxy (or 3-keto, 3-hydroxy), 4-methyl, 9,11-dihydroxy glycols (phthiocerols) and p-glycosylated phenylglycols (glycosyl phenolphthiocerols), respectively, that are diesterified with di-, tri-, and tetramethyl-branched acyl chains (mycocerosates) (reviewed in reference 28). PDIMs have been identified in Mycobacterium tuberculosis, M. africanum, M. bovis, M. leprae, M. marinum, M. ulcerans, M. kansasii, M. haemophilum, M. microti, and M. gastri, all of which are pathogenic for humans or animals. PGLs are produced by the same set of pathogenic mycobacterial species, except that in M. tuberculosis only a subset of clinical isolates produces PGLs.The role of PDIMs in virulence was first suggested by two independent studies using signature-tagged transposon mutagenesis, which identified mutants of M. tuberculosis that were unable to either produce or properly localize PDIMs to the cell wall and demonstrated that these mutants were attenuated in animal models of infection (8,12,33). Since then, circumstantial evidence supporting a role for PDIMs in M. tuberculosis virulence has accumulated. The role of PGLs in M. tuberculosis virulence is less clear and is confounded by the fact that laboratory strains (H37Rv, Erdman) and many clinical isolates, including CDC1551 and MT103, are naturally deficient in PGL production due to a 7-basepair deletion in pks15/1, while some clinical isolates of the East Asian lineage have an intact pks15/1 gene and produce PGLs (31). Mutations of pks15/1 in M. tuberculosis HN878, a strain that produces both PDIMs and PGLs and exhibits a hypervirulent phenotype in infect...

BackgroundGene amplification is a frequent manifestation of genomic instability that plays a role in tumour progression and development of drug resistance. It is manifested cytogenetically as extrachromosomal double minutes (DMs) or intrachromosomal homogeneously staining regions (HSRs). To better understand the molecular mechanism by which HSRs and DMs are formed and how they relate to the development of methotrexate (MTX) resistance, we used two model systems of MTX-resistant HT-29 colon cancer cell lines harbouring amplified DHFR primarily in (i) HSRs and (ii) DMs.ResultsIn DM-containing cells, we found increased expression of non-homologous end joining (NHEJ) proteins. Depletion or inhibition of DNA-PKcs, a key NHEJ protein, caused decreased DHFR amplification, disappearance of DMs, increased formation of micronuclei or nuclear buds, which correlated with the elimination of DHFR, and increased sensitivity to MTX. These findings indicate for the first time that NHEJ plays a specific role in DM formation, and that increased MTX sensitivity of DM-containing cells depleted of DNA-PKcs results from DHFR elimination. Conversely, in HSR-containing cells, we found no significant change in the expression of NHEJ proteins. Depletion of DNA-PKcs had no effect on DHFR amplification and resulted in only a modest increase in sensitivity to MTX. Interestingly, both DM-containing and HSR-containing cells exhibited decreased proliferation upon DNA-PKcs depletion.ConclusionsWe demonstrate a novel specific role for NHEJ in the formation of DMs, but not HSRs, in MTX-resistant cells, and that NHEJ may be targeted for the treatment of MTX-resistant colon cancer.

The proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) family proteins are prevalent in pathogenic mycobacteria and play a diverse role in mycobacterial pathogenesis. While some members have been studied, the function of most PE/PPE proteins remains unknown. In this study, we isolated a transposon-inactivated PPE38 mutant of Mycobacterium marinum and characterized its phenotype. We found that the PPE38 protein is associated with the cell wall and exposed on the cell surface. The inactivation of PPE38 altered the bacterial cell surface properties and led to deficiencies in cord formation, sliding motility, and biofilm formation. The PPE38 mutant was defective in phagocytosis by macrophages and exhibited reduced virulence in adult zebrafish. We also found that PPE38 is involved in the induction of proinflammatory cytokines in infected macrophages. Together, our results indicate that PPE38, a previously uncharacterized protein, plays a role in mycobacterial virulence, presumably by modulating the host innate immune response. Mycobacterium tuberculosis is a highly successful human pathogen that latently infects one-third of the world's population, causing 10 million new infections and 2 million deaths annually. One reason for the success of M. tuberculosis lies in its ability to evade host immune defense mechanisms and to create a niche within host cells, enabling the bacterium to persist for long periods. M. tuberculosis infects and survives within macrophages by evading macrophage killing mechanisms through a variety of strategies (reviewed in references 51 and 56). During persistent infection, M. tuberculosis is thought to reside within a granuloma, a cellular accumulation around the bacilli that is comprised mainly of macrophages, dendritic cells, T cells, B cells, and fibroblasts (reviewed in reference 31). Granulomas are generally thought to contain the infection by limiting bacterial growth and spread (21, 61). However, recent studies by Ramakrishnan and coworkers using zebrafish embryos demonstrated that the granuloma is a dynamic structure and that Mycobacterium marinum, an aquatic mycobacterium closely related to M. tuberculosis, uses the granuloma as a niche to recruit uninfected macrophages, raising the possibility that granulomas are exploited by the pathogen for expansion and dissemination in early infection (23,25,26).PE and PPE family proteins, named for the conserved N-terminal-domain-containing proline-glutamic acid (PE) motif or proline-proline-glutamine (PPE) motif, are unique to mycobacteria and particularly prevalent in pathogenic mycobacteria (35). They account for a significant fraction (ϳ10%) of the coding capacity of pathogenic mycobacteria. There are 168 PE/PPE proteins in M. tuberculosis (22) and 281 in M. marinum (64). The relatively conserved N termini are approximately 110 and 180 amino acids in the PE and PPE families, respectively. The C-terminal domains of both the PE and PPE protein families are highly variable in both size and sequence and often contain repetitive ...

We conducted a meta-analysis to assess the association between tumor necrosis factor-alpha (TNF-alpha) gene TNFA -308 (G>A), TNFA -238 (G>A), TNFA -857 (C>T), TNFA -863 (C>A), TNFA -1031 (T>C), TNFA -1210 (A>T) polymorphisms and breast cancer(BC) susceptibility. We also performed subgroup analyses based on ethnicity (Caucasian, Asian, and African). An extensive search was performed to identify all case-control studies investigating such association. Thirteen eligible studies, including 10,236 BC patients and 13,143 controls, were identified. No significant association was observed in all genotypes in worldwide populations, but stratification by ethnicity indicated that the TNFA -308 A allele was associated with a decreased risk of BC compared with the G allele in Caucasian individuals (OR = 0.927, 95%CI = 0.879-0.978). Similar results were obtained when the A/A +A/G genotype was compared with the G/G genotype. In addition, meta-analysis results indicated that the A/A genotype of TNFA -308 was a risk factor for BC in African (A/A vs. G/G OR = 4.085 95%CI = 1.460-11.425; A/A vs. G/A OR = 4.861 95%CI = 1.746-13.527; A/A vs. G/A + G/G OR = 4.246 95%CI = 1.551-11.625), but not in Caucasian or Asian individuals. In conclusion, the results of this meta-analysis indicate that the TNFA -308 A allele may be an important protective factor for BC in European individuals, but it is not likely to confer susceptibility to BC in worldwide populations. In addition, the AA genotype of TNFA -308 may be a risk factor for BC in African individuals. Besides, other polymorphisms were not associated with BC susceptibility.