Chlamydia muridarum infection of macrophages elicits bactericidal nitric oxide production via reactive oxygen species and cathepsin B

The ability of certain species of Chlamydia to inhibit the biogenesis of phagolysosomes permits their survival and replication within macrophages. The survival of macrophage-adapted chlamydiae correlates with the multiplicity of infection (MOI), and optimal chlamydial growth occurs in macrophages infected at an MOI of ≤1. In this study, we examined the replicative capacity of Chlamydia muridarum in the RAW 264.7 murine macrophage cell line at different MOIs. C. muridarum productively infected these macrophages at low MOIs but yielded few viable elementary bodies (EBs) when macrophages were infected at a moderate (10) or high (100) MOI. While high MOIs caused cytotoxicity and irreversible host cell death, macrophages infected at a moderate MOI did not show signs of cytotoxicity until late in the infectious cycle. Inhibition of host protein synthesis rescued C. muridarum in macrophages infected at a moderate MOI, implying that chlamydial growth was blocked by activated defense mechanisms. Conditioned medium from these macrophages was antichlamydial and contained elevated levels of interleukin 1β (IL-1β), IL-6, IL-10, and beta interferon (IFN-β). Macrophage activation depended on Toll-like receptor 2 (TLR2) signaling, and cytokine production required live, transcriptionally active chlamydiae. A hydroxyl radical scavenger and inhibitors of inducible nitric oxide synthase (iNOS) and cathepsin B also reversed chlamydial killing. High levels of reactive oxygen species (ROS) led to an increase in cathepsin B activity, and pharmacological inhibition of ROS and cathepsin B reduced iNOS expression. Our data demonstrate that MOI-dependent TLR2 activation of macrophages results in iNOS induction via a novel ROS- and cathepsin-dependent mechanism to facilitate C. muridarum clearance

Mutational Analysis of the Chlamydia muridarum Plasticity Zone

Pathogenically diverse Chlamydia spp. can have surprisingly similar genomes. C. trachomatis isolates that cause trachoma, sexually transmitted genital tract infections (chlamydia) and invasive lymphogranuloma venereum (LGV), and the murine strain C. muridarum share 99% of their gene content. A region of high genomic diversity between Chlamydia spp. termed the Plasticity Zone (PZ) may encode niche-specific virulence determinants that dictate pathogenic diversity. We hypothesized that PZ genes might mediate the greater virulence and IFN-γ resistance of C. muridarum compared to C. trachomatis in the murine genital tract. To test this hypothesis, we isolated and characterized a series of C. muridarum PZ nonsense mutants. Strains with nonsense mutations in chlamydial cytotoxins, guaBA-add and a phospholipase D homolog developed normally in cell culture. Two of the cytotoxin mutants were less cytotoxic than wild-type suggesting that the cytotoxins may be functional. However, none of the PZ nonsense mutants exhibited increased IFN-γ sensitivity in cell culture or were profoundly attenuated in a murine genital tract infection model. Our results suggest that C. muridarum PZ genes are transcribed and some may produce functional proteins, but are dispensable for infection of the murine genital tract

Evaluation of oral submucous fibrosis using ultrasonographic technique: A new diagnostic tool

Aim: The aim of this study is to evaluate oral submucous fibrosis (OSMF) by clinical and histopathological examination, and compare the results with those from ultrasonographic technique. Materials and Methods: 30 clinically diagnosed OSMF patients were subjected to both ultrasonographic and histopathological evaluation before treatment. Later, only ultrasonographical examination was done during 4 th and 8 th week of treatment. Prognosis of the lesion for the treatment was evaluated. Peak systolic velocity (PSV) of blood in the lesional area was statistically analyzed. 10 normal individuals without any mucosal lesions were considered as the control group. Results: In normal individuals, ultrasonography delineates normal mucosa with uniform fine mottled appearance with interspersed hypoechoic areas. Color Doppler and spectral Doppler depicts uniform distribution of blood vessels and normal peak systolic velocity of blood respectively. All OSMF patients were diagnosed upon clinical and histopathological examination. Clinical examination revealed 14 individuals with unilateral palpable fibrotic bands and 16 individuals with bilateral fibrotic bands whereas, ultrasonographic evaluation revealed 6 individuals with unilateral fibrotic bands and 24 individuals were with bilateral fibrotic bands, which was statistically significant. Ultrasonography demonstrated number, length and thickness of the fibrotic bands. Color Doppler and spectral Doppler showed decreased vascularity and PSV in lesional area. Prognosis evaluation revealed 25 cases of good prognosis and 5 cases were showed poor prognosis. Wilcoxon Signed Ranks Test revealed no significant difference of PSV was seen in poor prognosis patients. Conclusion: Ultrasonography is a non-invasive diagnostic tool for OSMF. It could be a better diagnostic tool compared to clinical and histopathological examination

Probiotic potential of lactic acid bacteria present in home made curd in southern India

Background & objectives: The human gut microbiota play a significant role in nutritional processes. The concept of probiotics has led to widespread consumption of food preparations containing probiotic microbes such as curd and yogurt. Curd prepared at home is consumed every day in most homes in southern India. In this study the home-made curd was evaluated for lactic acid bacteria (LAB) with probiotic potential. Methods: Fifteen LAB (12 lactobacilli, 1 l0 actococcus , 2 Leuconostoc) and one yeast isolated from home-made curd were evaluated for resistance to acid, pepsin, pancreatin and bile salts; antimicrobial resistance; intrinsic antimicrobial activity; adherence to Caco-2 epithelial cells; ability to block pathogen adherence to Caco-2 cells; ability to inhibit interleukin (IL)-8 secretion from HT-29 epithelial cells in response to Vibrio cholerae; and ability to induce anti-inflammatory cytokine expression in THP-1 monocyte cells. Results: Lactobacillus abundance in fermenting curd peaked sharply at 12 h. Nine of the strains survived exposure to acid (pH 3.0) for at least one hour, and all strains survived in the presence of pancreatin or bile salts for 3 h. None showed haemolytic activity. All were resistant to most antimicrobials tested, but were sensitive to imipenem. Most strains inhibited the growth of Salmonella Typhimurium while five inhibited growth of V. cholerae O139. Seven strains showed adherence to Caco-2 cells ranging from 20-104 per cent of adherence of an adherent strain of Escherichia coli, but all inhibited V. cholerae adherence to Caco-2 cells by 20-100 per cent. They inhibited interleukin-8 secretion from HT-29 cells, in response to V. cholerae, by 50-80 per cent. Two strains induced IL-10 and IL-12 messenger ribonucleic acid (mRNA) expression in THP-1 cells. Interpretation & conclusions: LAB in curd had properties consistent with probiotic potential, but these were not consistent across species. LAB abundance in curd increased rapidly at 12 h of fermentation at room temperature and declined thereafter

A mevalonate bypass system facilitates elucidation of plastid biology in malaria parasites.

Malaria parasites rely on a plastid organelle for survival during the blood stages of infection. However, the entire organelle is dispensable as long as the isoprenoid precursor, isopentenyl pyrophosphate (IPP), is supplemented in the culture medium. We engineered parasites to produce isoprenoid precursors from a mevalonate-dependent pathway, creating a parasite line that replicates normally after the loss of the apicoplast organelle. We show that carbon-labeled mevalonate is specifically incorporated into isoprenoid products, opening new avenues for researching this essential class of metabolites in malaria parasites. We also show that essential apicoplast proteins, such as the enzyme target of the drug fosmidomycin, can be deleted in this mevalonate bypass parasite line, providing a new method to determine the roles of other important apicoplast-resident proteins. Several antibacterial drugs kill malaria parasites by targeting basic processes, such as transcription, in the organelle. We used metabolomic and transcriptomic methods to characterize parasite metabolism after azithromycin treatment triggered loss of the apicoplast and found that parasite metabolism and the production of apicoplast proteins is largely unaltered. These results provide insight into the effects of apicoplast-disrupting drugs, several of which have been used to treat malaria infections in humans. Overall, the mevalonate bypass system provides a way to probe essential aspects of apicoplast biology and study the effects of drugs that target apicoplast processes

The Minimal Unit Of Infection: Mycobacterium Tuberculosis In The Macrophage

The interaction between Mycobacterium tuberculosis and its host cell is highly complex and extremely intimate. Were it not for the disease, one might regard this interaction at the cellular level as an almost symbiotic one. The metabolic activity and physiology of both cells are shaped by this coexistence. We believe that where this appreciation has greatest significance is in the field of drug discovery. Evolution rewards efficiency, and recent data from many groups discussed in this review indicate that M. tuberculosis has evolved to utilize the environmental cues within its host to control large genetic programs or regulons. But these regulons may represent chinks in the bacterium\u27s armor because they include off-target effects, such as the constraint of the metabolic plasticity of M. tuberculosis. A prime example is how the presence of cholesterol within the host cell appears to limit the ability of M. tuberculosis to fully utilize or assimilate other carbon sources. And that is the reason for the title of this review. We believe firmly that, to understand the physiology of M. tuberculosis and to identify new drug targets, it is imperative that the bacterium be interrogated within the context of its host cell. The constraints induced by the environmental cues present within the host cell need to be preserved and exploited. The M. tuberculosis-infected macrophage truly is the minimal unit of infection

The Minimal Unit of Infection: Mycobacterium tuberculosis

The interaction between Mycobacterium tuberculosis and its host cell is highly complex and extremely intimate. Were it not for the disease, one might regard this interaction at the cellular level as an almost symbiotic one. The metabolic activity and physiology of both cells are shaped by this coexistence. We believe that where this appreciation has greatest significance is in the field of drug discovery. Evolution rewards efficiency, and recent data from many groups discussed in this review indicate that M. tuberculosis has evolved to utilize the environmental cues within its host to control large genetic programs or regulons. But these regulons may represent chinks in the bacterium\u27s armor because they include off-target effects, such as the constraint of the metabolic plasticity of M. tuberculosis. A prime example is how the presence of cholesterol within the host cell appears to limit the ability of M. tuberculosis to fully utilize or assimilate other carbon sources. And that is the reason for the title of this review. We believe firmly that, to understand the physiology of M. tuberculosis and to identify new drug targets, it is imperative that the bacterium be interrogated within the context of its host cell. The constraints induced by the environmental cues present within the host cell need to be preserved and exploited. The M. tuberculosis-infected macrophage truly is the minimal unit of infection