Lisa Y. Armitige

Processing and Presentation of a Mycobacterial Antigen 85B Epitope by Murine Macrophages Is Dependent on the Phagosomal Acquisition of Vacuolar Proton ATPase and In Situ Activation of Cathepsin D

Mycobacterium tuberculosis (strain H37Rv) and bacillus Calmette-Guérin (BCG) vaccine inhibit phagosome maturation in macrophages and their effect on processing, and presentation of a secreted Ag85 complex B protein, Ag85B, by mouse macrophages was analyzed. Macrophages were infected with GFP-expressing mycobacterial strains and analyzed for in situ localization of vacuolar proton ATPase (v-ATPase) and cathepsin D (Cat D) using Western blot analysis and immunofluorescence. H37Rv and BCG phagosomes excluded the v-ATPase and maintained neutral pH while the attenuated H37Ra strain acquired v-ATPase and acidified. Mycobacterial phagosomes acquired Cat D, although strains BCG and H37Rv phagosomes contained the inactive 46-kDa form, whereas H37Ra phagosomes had the active 30-kDa form. Infected macrophages were overlaid with a T cell hybridoma specific for an Ag85B epitope complexed with MHC class II. Coincident with active Cat D, H37Ra-infected macrophages presented the epitope to T cells inducing IL-2, whereas H37Rv- and BCG-infected macrophages were less efficient in IL-2 induction. Bafilomycin inhibited the induction of macrophage-induced IL-2 from T cells indicating that v-ATPase was essential for macrophage processing of Ag85B. Furthermore, the small interfering RNA interference of Cat D synthesis resulted in a marked decrease in the levels of macrophage-induced IL-2. Thus, a v-ATPase-dependent phagosomal activation of Cat D was required for the generation of an Ag85B epitope by macrophages. Reduced processing of Ag85B by H37Rv- and BCG-infected macrophages suggests that phagosome maturation arrest interferes with the efficient processing of Ags in macrophages. Because Ag85B is immunodominant, this state may lead to a decreased ability of the wild-type as well as the BCG vaccine to induce protective immunity.

The ΔfbpA mutant derived from Mycobacterium tuberculosis H37Rv has an enhanced susceptibility to intracellular antimicrobial oxidative mechanisms, undergoes limited phagosome maturation and activates macrophages and dendritic cells

Mycobacterium tuberculosis H37Rv (Mtb) excludes phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) while preventing lysosomal fusion in macrophages (MΦs). The antigen 85A deficient (ΔfbpA) mutant of Mtb was vaccinogenic in mice and the mechanisms of attenuation were compared with MΦs infected with H37Rv and BCG. ΔfbpA contained reduced amounts of trehalose 6, 6, dimycolate and induced minimal levels of SOCS‐1 in MΦs. Blockade of oxidants enhanced the growth of ΔfbpA in MΦs that correlated with increased colocalization with phox and iNOS. Green fluorescent protein‐expressing strains within MΦs or purified phagosomes were analysed for endosomal traffick with immunofluorescence and Western blot. ΔfbpA phagosomes were enriched for rab5, rab11, LAMP‐1 and Hck suggesting enhanced fusion with early, recycling and late endosomes in MΦs compared with BCG or H37Rv. ΔfbpA phagosomes were thus more mature than H37Rv or BCG although, they failed to acquire rab7 and CD63 preventing lysosomal fusion. Finally, ΔfbpA infected MΦs and dendritic cells (DCs) showed an enhanced MHC‐II and CD1d expression and primed immune T cells to release more IFN‐γ compared with those infected with BCG and H37Rv. ΔfbpA was thus more immunogenic in MΦs and DCs because of an enhanced susceptibility to oxidants and increased maturation.

A Novel Single-Nucleotide Polymorphism in the Lactoferrin Gene Is Associated with Susceptibility to Diarrhea in North American Travelers to Mexico

BACKGROUND Diarrhea affects 40%-60% of travelers from industrialized nations who visit developing countries and is due to bacterial, viral, and parasitic agents. Lactoferrin is bactericidal to enteric pathogens, modulates the intestinal immune response, and is excreted in stool in response to infection with intestinal organisms. We investigated the impact that selected single-nucleotide polymorphisms (SNPs) in the human lactoferrin gene have on susceptibility to travelers diarrhea. METHODS Adults who had recently arrived in Mexico were studied prospectively for the occurrence and causal agent(s) of travelers diarrhea, and genotyping was performed for 9 distinct lactoferrin SNPs. RESULTS Of the 9 SNPs studied, only 1 SNP (located in exon 15) was associated with travelers diarrhea (P=.004). When compared with healthy travelers, and after adjustment for known risk factors for travelers diarrhea (such as age and duration and season of travel), subjects with the T/T genotype in amino acid position 632 were more likely to develop travelers diarrhea (67% vs. 33%; relative risk [RR], 1.4; 95% CI, 1.2-1.7; P<.001), to have diarrhea with a pathogen identified (RR, 1.3; 95% CI, 1.1-1.6; P=.03), and to have a marker of intestinal inflammation in stool specimens (blood, mucus, or white blood cells; 52% vs. 38%; P=.036). The association was also significant when norovirus was not identified in stool samples (RR, 1.34; 95% CI, 1.06-1.34; P=.01). CONCLUSIONS The T/T genotype in position codon 632 of the lactoferrin gene is associated with susceptibility to diarrhea in North Americans traveling to Mexico.

A mutant of Mycobacterium tuberculosis H37Rv that lacks expression of antigen 85A is attenuated in mice but retains vaccinogenic potential.

ABSTRACT The fbpA and fbpB genes encoding the 85A and 85B proteins of Mycobacterium tuberculosis H37Rv, respectively, were disrupted, the mutants were examined for their ability to survive, and the strain lacking 85A (ΔfbpA) was tested for its ability to immunize mice. The ΔfbpA mutant was attenuated in mice after intravenous or aerosol infection, while replication of the ΔfbpB mutant was similar to that of the wild type. Complementation of the fbpA gene in ΔfbpA restored its ability to grow in the lungs of mice. The ΔfbpA mutant induced a stronger expression of pulmonary mRNA messages in mice for tumor necrosis factor alpha, interleukin-1 beta (IL-1β), gamma interferon, IL-6, IL-2, and inducible nitric oxide (NO) synthase, which led to its decline, while H37Rv persisted despite strong immune responses. H37Rv and ΔfbpA both induced NO in macrophages and were equally susceptible to NO donors, although ΔfbpA was more susceptible in vitro to peroxynitrite and its growth was enhanced by NO inhibitors in mice and macrophages. Aerosol-infected mice, which cleared a low-dose ΔfbpA infection, resisted a challenge with virulent M. tuberculosis. Mice subcutaneously immunized with ΔfbpA or Mycobacterium bovis BCG and challenged with M. tuberculosis also showed similar levels of protection, marked by a reduction in the growth of challenged M. tuberculosis. The ΔfbpA mutant was thus attenuated, unlike ΔfbpB, but was also vaccinogenic against tuberculosis. Attenuation was incomplete, however, since ΔfbpA revived in normal mice after 370 days, suggesting that revival was due to immunosenescence but not compensation by the fbpB or fbpC gene. Antigen 85A thus affects susceptibility to peroxynitrite in M. tuberculosis and appears to be necessary for its optimal growth in mice.

TB Research at UT-Houston – A review of cord factor: new approaches to drugs, vaccines and the pathogenesis of tuberculosis

Tuberculosis remains a major threat as drug resistance continues to increase. Pulmonary tuberculosis in adults is responsible for 80% of clinical cases and nearly 100% of transmission of infection. Unfortunately, since we have no animal models of adult type pulmonary tuberculosis, the most important type of disease remains largely out of reach of modern science and many fundamental questions remain unanswered. This paper reviews research dating back to the 1950s providing compelling evidence that cord factor (trehalose 6,6 dimycolate [TDM]) is essential for understanding tuberculosis. However, the original papers by Bloch and Noll were too far ahead of their time to have immediate impact. We can now recognize that the physical and biologic properties of cord factor are unprecedented in science, especially its ability to switch between two sets of biologic activities with changes in conformation. While TDM remains on organisms, it protects them from killing within macrophages, reduces antibiotic effectiveness and inhibits the stimulation of protective immune responses. If it comes off organisms and associates with lipid, TDM becomes a driver of tissue damage and necrosis. Studies emanating from cord factor research have produced (1) a rationale for improving vaccines, (2) an approach to new drugs that overcome natural resistance to antibiotics, (3) models of caseating granulomas that reproduce multiple manifestations of human tuberculosis. (4) evidence that TDM is a key T cell antigen in destructive lesions of tuberculosis, and (5) a new understanding of the pathology and pathogenesis of postprimary tuberculosis that can guide more informative studies of long standing mysteries of tuberculosis.

Influence of Host Interleukin-10 Polymorphisms on Development of Traveler's Diarrhea Due to Heat-Labile Enterotoxin-Producing Escherichia coli in Travelers from the United States Who Are Visiting Mexico

ABSTRACT Up to 60% of U.S. visitors to Mexico develop travelers diarrhea (TD), mostly due to enterotoxigenic Escherichia coli (ETEC) strains that produce heat-labile (LT) and/or heat-stable (ST) enterotoxins. Distinct single-nucleotide polymorphisms (SNPs) within the interleukin-10 (IL-10) promoter have been associated with high, intermediate, or low production of IL-10. We conducted a prospective study to investigate the association of SNPs in the IL-10 promoter and the occurrence of TD in ETEC LT-exposed travelers. Sera from U.S. travelers to Mexico collected on arrival and departure were studied for ETEC LT seroconversion by using cholera toxin as the antigen. Pyrosequencing was performed to genotype IL-10 SNPs. Stools from subjects who developed diarrhea were also studied for other enteropathogens. One hundred twenty-one of 569 (21.3%) travelers seroconverted to ETEC LT, and among them 75 (62%) developed diarrhea. Symptomatic seroconversion was more commonly seen in subjects who carried a genotype producing high levels of IL-10; it was seen in 83% of subjects with the GG genotype versus 54% of subjects with the AA genotype at IL-10 gene position −1082 (P, 0.02), in 71% of those with the CC genotype versus 33% of those with the TT genotype at position −819 (P, 0.005), and in 71% of those with the CC genotype versus 38% of those with the AA genotype at position −592 (P, 0.02). Travelers with the GCC haplotype were more likely to have symptomatic seroconversion than those with the ATA haplotype (71% versus 38%; P, 0.002). Travelers genetically predisposed to produce high levels of IL-10 were more likely to experience symptomatic ETEC TD.

Determination of Vancomycin and Daptomycin MICs by Different Testing Methods for Methicillin-Resistant Staphylococcus aureus

ABSTRACT Vancomycin and daptomycin MICs from 161 isolates of methicillin-resistant Staphylococcus aureus (MRSA) were compared using commercial and in-house broth microdilution, Etest, and common automated methods. Vancomycin Etest MICs were higher than those of other methods, whereas the MICs for daptomycin testing were comparable. Vancomycin MICs vary depending on the testing methodology.

A review of tuberculosis: reflections on the past, present and future of a global epidemic disease.

Tuberculosis remains the leading cause of death worldwide from a single infectious organism. Approximately 32% of the world’s population is infected and an estimatedtwo million people die annually from this treatable disease. Over the past 50 years, with medical treatment and standard public health practices, tuberculosis diminished in developed countries and resulted in a loss of interest and funding for research in improving diagnostic and treatment options. In developing countries, efforts including BCG vaccination have failed to control tuberculosis and the disease continues to spread as the world becomes more globalized. At the same time, multidrug resistant tuberculosis has emerged, challenging even the most advance treatment centers. Better diagnostic techniques, control measures and treatment options are desperately needed but advances require worldwide commitment to battle this age-old disease.

The fbpA/sapM Double Knock Out Strain of Mycobacterium tuberculosis Is Highly Attenuated and Immunogenic in Macrophages

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death due to bacterial infections in mankind, and BCG, an attenuated strain of Mycobacterium bovis, is an approved vaccine. BCG sequesters in immature phagosomes of antigen presenting cells (APCs), which do not fuse with lysosomes, leading to decreased antigen processing and reduced Th1 responses. However, an Mtb derived ΔfbpA attenuated mutant underwent limited phagosome maturation, enhanced immunogenicity and was as effective as BCG in protecting mice against TB. To facilitate phagosome maturation of ΔfbpA, we disrupted an additional gene sapM, which encodes for an acid phosphatase. Compared to the wild type Mtb, the ΔfbpAΔsapM (double knock out; DKO) strain was attenuated for growth in mouse macrophages and PMA activated human THP1 macrophages. Attenuation correlated with increased oxidants in macrophages in response to DKO infection and enhanced labeling of lysosomal markers (CD63 and rab7) on DKO phagosomes. An in vitro Antigen 85B peptide presentation assay was used to determine antigen presentation to T cells by APCs infected with DKO or other mycobacterial strains. This revealed that DKO infected APCs showed the strongest ability to present Ag85B to T cells (>2500 pgs/mL in 4 hrs) as compared to APCs infected with wild type Mtb or ΔfbpA or ΔsapM strain (<1000 pgs/mL in 4 hrs), indicating that DKO strain has enhanced immunogenicity than other strains. The ability of DKO to undergo lysosomal fusion and vacuolar acidification correlated with antigen presentation since bafilomycin, that inhibits acidification in APCs, reduced antigen presentation. Finally, the DKO vaccine elicited a better Th1 response in mice after subcutaneous vaccination than either ΔfbpA or ΔsapM. Since ΔfbpA has been used in mice as a candidate vaccine and the DKO (ΔfbpAΔsapM) mutant is more immunogenic than ΔfbpA, we propose the DKO is a potential anti-tuberculosis vaccine.

Predictors of Relapse of Methicillin-Resistant Staphylococcus aureus Bacteremia after Treatment with Vancomycin

ABSTRACT The risk factors for relapse of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia after vancomycin treatment are unknown. Diversilab typing was used to classify recurrent bacteremia as relapse or reinfection. Bacteremia for >7 days and staphylococcal cassette chromosome mec element (SCCmec) type II were independently associated with relapse of MRSA bacteremia after vancomycin treatment.