The Centers for Disease Control and Prevention and National Institutes of Health convened a multidisciplinary meeting to discuss surrogate markers of treatment response in tuberculosis. The goals were to assess recent surrogate marker research and to provide specific recommendations for (1) the qualification and validation of biomarkers of treatment outcome; (2) the standardization of specimen and data collection for future clinical trials, including a minimum set of samples and collection time points; and (3) the creation of a specimen repository to support biomarker testing. This article summarizes these recommendations and provides a roadmap for their implementation.

The study of infectious agents, their pathogenesis, the host response and the evaluation of newly developed countermeasures often requires the use of a living system. Murine models are frequently used to undertake such investigations with the caveat that non-biased measurements to assess the progression of infection are underutilized. Instead, murine models predominantly rely on symptomology exhibited by the animal to evaluate the state of the animal's health and to determine when euthanasia should be performed. In this study, we used subcutaneous temperature as a non-subjective measurement to follow and compare infection in mice inoculated with Francisella tularensis, a Gram-negative pathogen that produces an acute and fatal illness in mice. A reproducible temperature pattern defined by three temperature phases (normal, febrile and hypothermic) was identified in all mice infected with F. tularensis, regardless of the infecting strain. More importantly and for the first time a non-subjective, ethical, and easily determined surrogate endpoint for death based on a temperature, termed drop point, was identified and validated with statistical models. In comparative survival curve analyses for F. tularensis strains with differing virulence, the drop point temperature yielded the same results as those obtained using observed time to death. Incorporation of temperature measurements to evaluate F. tularensis was standardized based on statistical models to provide a new level of robustness for comparative analyses in mice. These findings should be generally applicable to other pathogens that produce acute febrile disease in animal models and offers an important tool for understanding and following the infection process.

One of the most effective and widely used antituberculosis (anti-TB) drugs is isoniazid (INH), a prodrug activated via oxidation that forms an adduct with NAD+ to inhibit NADH-dependent targets of Mycobacterium tuberculosis, such as enoyl-acyl carrier protein reductase (InhA). The metabolic by-products and potentially toxic intermediates resulting from INH therapy have been identified through a large body of work. However, an INH-NAD adduct or structures related to this adduct have not been identified in specimens from human TB patients or animal models of TB. Analyses by mass spectrometry of urine collected from TB patients in a study conducted by the NIAID-funded Tuberculosis Research Unit identified 4-isonicotinoylnicotinamide (C12H9N3O2) as a novel metabolite of INH therapy. This compound was formed by M. tuberculosis strains in a KatG-dependent manner but could also be produced by mice treated with INH independent of an M. tuberculosis infection. Thus, the 4-isonicotinoylnicotinamide observed in human urine samples is likely derived from the degradation of oxidized INH-NAD adducts and provides direct evidence of host INH activation.

A method is described using Desorption Electrospray Ionization (DESI) Mass Spectrometry (MS) to obtain phospholipid mass spectral profiles from crude lung tissue extracts. The measured DESI-mass spectral lipid fingerprints were then analyzed by unsupervised learning principal components analysis (PCA). This combined approach was used to differentiate the effect(s) of two vaccination routes on lipid composition in mice lung. Specifically, the two vaccination routes compared were intra-nasal (i.n.) and intra-dermal (i.d.) inoculation of the Francisella tularensis Live Vaccine Strain (Ft-LVS). Lung samples of control and LVS-inoculated mice were quickly extracted with a methanol/chloroform solution and the crude extract directly analyzed by DESI-MS, with a total turnaround time of less than 10 min per sample. All the measured DESI-mass spectra (in both positive and negative ion mode) were compared via PCA which resulted in clear differentiation of mass spectral profiles of i.n.-inoculated mice lung tissues from those of i.d.-inoculated and control mice lung tissues. Lipid biomarkers responsible for sample differentiation were identified via tandem MS (MS/MS) measurements or by comparison with mass spectra of lipid standards. The DESI-MS approach here described provided a practical and rapid means to analyze tissue samples without extensive extractions and solvent changes.

The molecular basis of pathogen-induced host cell apoptosis is well characterized for a number of microorganisms. Mycobacterium tuberculosis is known to induce apoptosis and it was shown that live but not heat killed M. tuberculosis stimulates this biological pathway in monocytes. The dependence of this activity on live bacilli led us to hypothesize that products released or secreted by M. tuberculosis are the primary apoptotic factors for human monocytes. Thus, the culture filtrate of in vitro grown M. tuberculosis strain H37Rv was fractioned by conventional chromatography and the apoptosis-inducing activity of individual fractions was measured on human monocytes. The tests employed included measurement of cell membrane damage, caspase activation, and cytokine release. Small molecular weight RNAs of M. tuberculosis were recognized as the predominant apoptosis inducing factors. The RNA was comprised primarily of tRNA and rRNA fragments that stably accumulate in the culture filtrate during early log-phase growth. The RNA fragments signaled through a caspase-8 dependent, caspase-1 and TNF-α independent pathway that ultimately compromised the human monocytes' ability to control M. tuberculosis infection. These studies provide the first report of bacterial RNA inducing apoptosis. They also provide a foundation to pursue pathways for secretion or release of nucleic acids from M. tuberculosis and the impact of secreted RNA fragments on pathogenesis.

Genotyping of Francisella tularensis (A1a, A1b, A2, and type B) and Francisella novicida has identified multiple differences between species and among F. tularensis subspecies and subpopulations. Variations in virulence, geographic distribution, and ecology are also known to exist among this group of bacteria, despite the >95% nucleotide identity in their genomes. This study expands the description of phenotypic differences by evaluating the ability of F. tularensis and F. novicida to degrade chitin analogs and produce active chitinases. Endochitinase activities were observed to vary among F. tularensis and F. novicida strains. The activity observed for F. tularensis strains was predominantly associated with whole-cell lysates, while the chitinase activity of F. novicida localized to the culture supernatant. In addition, the overall level of chitinase activity differed among the subpopulations of F. tularensis and between the species. Bioinformatic analyses identified two new putative chitinase genes (chiC and chiD), as well as the previously described chiA and chiB. However, the presence of these four open reading frames as intact genes or pseudogenes was found to differ between Francisella species and F. tularensis subspecies and subpopulations. Recombinant production of the putative chitinases and enzymatic evaluations revealed ChiA, ChiB, ChiC, and ChiD possessed dissimilar chitinase activities. These biochemical studies coupled with bioinformatic analyses and the evaluation of chiA and chiC knockouts in F. tularensis A1 and A2 strains, respectively, provided a molecular basis to explain the differential chitinase activities observed among the species and subpopulations of Francisella.

Protection against virulent pathogens that cause acute, fatal disease is often hampered by development of microbial resistance to traditional chemotherapeutics. Further, most successful pathogens possess an array of immune evasion strategies to avoid detection and elimination by the host. Development of novel, immunomodulatory prophylaxes that target the host immune system, rather than the invading microbe, could serve as effective alternatives to traditional chemotherapies. Here we describe the development and mechanism of a novel pan-anti-bacterial prophylaxis. Using cationic liposome non-coding DNA complexes (CLDC) mixed with crude F. tularensis membrane protein fractions (MPF), we demonstrate control of virulent F. tularensis infection in vitro and in vivo. CLDC+MPF inhibited bacterial replication in primary human and murine macrophages in vitro. Control of infection in macrophages was mediated by both reactive nitrogen species (RNS) and reactive oxygen species (ROS) in mouse cells, and ROS in human cells. Importantly, mice treated with CLDC+MPF 3 days prior to challenge survived lethal intranasal infection with virulent F. tularensis. Similarly to in vitro observations, in vivo protection was dependent on the presence of RNS and ROS. Lastly, CLDC+MPF was also effective at controlling infections with Yersinia pestis, Burkholderia pseudomallei and Brucella abortus. Thus, CLDC+MPF represents a novel prophylaxis to protect against multiple, highly virulent pathogens.

Author Summary

Conventional treatment of bacterial infections typically includes administration of antibiotics. However, many pathogens have developed spontaneous resistance to commonly used antibiotics. Development of new compounds that stimulate the host immune system to directly kill bacteria by mechanisms different from those utilized by antibiotics may serve as effective alternatives to antibiotic therapy. In this report, we describe a novel compound capable of controlling infections mediated by different, unrelated bacteria via the induction of host derived reactive oxygen and reactive nitrogen species. This compound is comprised of cationic liposome DNA complexes (CLDC) and crude membrane preparations (MPF) obtained from attenuated Francisella tularensis Live Vaccine Strain (LVS). Pretreatment of primary mouse or human cells limited replication of virulent F. tularensis, Burkholderia pseudomallei, Yersinia pestis and Brucella abortus in vitro. CLDC+MPF was also effective for controlling lethal pulmonary infections with virulent F. tularensis. Thus, CLDC+MPF represents a novel antimicrobial for treatment of lethal, acute, bacterial infections.

The sequencing of the Mycobacterium tuberculosis genome revealed the existence of several genes encoding novel proteins with unknown functions, one of which is the proline-threonine repetitive protein (PTRP; Rv0538). Genomic studies of various mycobacterial species and M. tuberculosis clinical isolates demonstrate that ptrp is specific to the M. tuberculosis complex and ubiquitous in clinical isolates. Enzyme-linked immunosorbent assay, Western blot analysis, and electron microscopic evaluation of M. tuberculosis subcellular fractions and intact bacteria confirm that PTRP is a cell wall protein. Antibodies to PTRP are present in serum specimens from human immunodeficiency virus (HIV)–negative, tuberculosis (TB)–positive and HIV-positive, TB-positive patients but not purified protein derivative (PPD)–negative or PPD-positive healthy control subjects, demonstrating its diagnostic potential. Epitope mapping of PTRP delineated 4 peptides that can identify >80% of sputum smear–positive and >50% of smear-negative, HIV-negative, TB-positive patients and >80% of HIV-positive, TB-positive patients. These results demonstrate that immunodominant epitopes of carefully selected M. tuberculosis–specific proteins can be used to devise a simple peptide-based serodiagnostic test for TB.

Objectives

Universally conserved events in cell division provide the opportunity for the development of novel chemotherapeutics against Mycobacterium tuberculosis. The aim of this study was to use the β-lactam antimicrobials cefalexin and piperacillin to inhibit FtsI and characterize the morphological changes and global transcriptional activities of genes to identify a signature response to FtsI inactivation.

Methods

Cefalexin and piperacillin were used to block cell division, and microscopy was used to evaluate the effects on bacterial morphology and ultrastructure. Global transcriptional analysis was performed to determine the impact of FtsI inhibition on cell cycle processes and to identify molecular markers.

Results

Inhibition of FtsI with cefalexin and piperacillin resulted in filamentous cells with multiple concentric rings and occasional branching as visualized by light and electron microscopy. Whole genome microarray-based transcriptional profiling and transcriptional mapping allowed the evaluation of cell cycle processes in response to inhibition of FtsI and characterization of transcriptional response and cell cycle processes.

Conclusions

This study substantiated that FtsZ-ring constriction and septal resolution require the transpeptidase activity of FtsI, making FtsI essential for cell division in M. tuberculosis. Therefore, FtsI is a target for drug discovery, and these studies provided a molecular signature of FtsI inactivation that can be applied to screening strategies for novel FtsI inhibitors.

SodC is one of two superoxide dismutases produced by Mycobacterium tuberculosis. This protein was previously shown to contribute to virulence and to act as a B-cell antigen. SodC is also a putative lipoprotein, and like other Sec-translocated mycobacterial proteins it was suggested to be modified with glycosyl units. To definitively define the glycosylation of SodC, we applied an approach that combined site-directed mutagenesis, lectin binding, and mass spectrometry. This resulted in identification of six O-glycosylated residues within a 13-amino-acid region near the N-terminus. Each residue was modified with one to three hexose units, and the most dominant SodC glycoform was modified with nine hexose units. In addition to O-glycosylation of threonine residues, this study provides the first evidence of serine O-glycosylation in mycobacteria. When combined with bioinformatic analyses, the clustering of O-glycosylation appeared to occur in a region of SodC with a disordered structure and not in regions important to the enzymatic activity of SodC. The use of recombinant amino acid substitutions to alter glycosylation sites provided further evidence that glycosylation influences proteolytic processing and ultimately positioning of cell wall proteins.

Objectives

Household contacts (HHCs) of pulmonary tuberculosis patients are at high risk of Mycobacterium tuberculosis infection and early disease development. Identification of individuals at risk of tuberculosis disease is a desirable goal for tuberculosis control. Interferon-gamma release assays (IGRAs) using specific M. tuberculosis antigens provide an alternative to tuberculin skin testing (TST) for infection detection. Additionally, the levels of IFNγ produced in response to these antigens may have prognostic value. We estimated the prevalence of M. tuberculosis infection by IGRA and TST in HHCs and their source population (SP), and assessed whether IFNγ levels in HHCs correlate with tuberculosis development.

Methods

A cohort of 2060 HHCs was followed for 2–3 years after exposure to a tuberculosis case. Besides TST, IFNγ responses to mycobacterial antigens: CFP, CFP-10, HspX and Ag85A were assessed in 7-days whole blood cultures and compared to 766 individuals from the SP in Medellín, Colombia. Isoniazid prophylaxis was not offered to child contacts because Colombian tuberculosis regulations consider it only in children under 5 years, TST positive without BCG vaccination.

Results

Using TST 65.9% of HHCs and 42.7% subjects from the SP were positive (OR 2.60, p<0.0001). IFNγ response to CFP-10, a biomarker of M. tuberculosis infection, tested positive in 66.3% HHCs and 24.3% from the SP (OR = 6.07, p<0.0001). Tuberculosis incidence rate was 7.0/1000 person years. Children <5 years accounted for 21.6% of incident cases. No significant difference was found between positive and negative IFNγ responders to CFP-10 (HR 1.82 95% CI 0.79–4.20 p = 0.16). However, a significant trend for tuberculosis development amongst high HHC IFNγ producers was observed (trend Log rank p = 0.007).

Discussion

CFP-10-induced IFNγ production is useful to establish tuberculosis infection prevalence amongst HHC and identify those at highest risk of disease. The high tuberculosis incidence amongst children supports administration of chemoprohylaxis to child contacts regardless of BCG vaccination.

CD4+ T cell clones derived from a leprosy lesion and patient blood were used to monitor the isolation and identification of an antigen associated with the self-limited form of the disease. Biochemical purification and genetic analysis identified the T cell antigen as a conserved mycobacterial lipoglycoprotein LprG. LprG-mediated activation of CD4+T cells required specific MHC class II restriction molecules and intracellular processing. Although LprG activated TLR2, this alone was not sufficient to stimulate nor did it inhibit T cell activation. A striking finding was that the carbohydrate moieties of LprG were required for optimal T cell activation, since recombinant LprG produced in Escherichia coli, or recombinant LprG produced in Mycobacterium smegmatis and digested by α-mannosidase did not activate T cells. This study demonstrates that the universe of bacterial T cell antigens includes lipoglycoproteins, which act as TLR2 ligands but also require glycosylation for MHC class II-restricted T cell activation in vivo.

Tularemia is caused by two subspecies of Francisella tularensis, F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). F. tularensis subsp. tularensis is further subdivided into two genetically distinct populations (A.I and A.II) that differ with respect to geographical location, anatomical source of recovered isolates, and disease outcome. Using two human clinical isolates, suppression subtractive hybridization was performed to identify 13 genomic regions of difference between A.I and A.II strains. Two PCR assays, one to identify A.I and A.II as well as to discriminate between F. tularensis subsp. holarctica and F. novicida and another specific for A.I, were developed. This is the first report to identify and characterize conserved genomic differences between A.I and A.II.

Background

Simple biomarkers are required to identify TB in both HIV−TB+ and HIV+TB+ patients. Earlier studies have identified the M. tuberculosis Malate Synthase (MS) and MPT51 as immunodominant antigens in TB patients. One goal of these investigations was to evaluate the sensitivity and specificity of anti-MS and –MPT51 antibodies as biomarkers for TB in HIV−TB+ and HIV+TB+ patients from a TB-endemic setting. Earlier studies also demonstrated the presence of these biomarkers during incipient subclinical TB. If these biomarkers correlate with incipient TB, their prevalence should be higher in asymptomatic HIV+ subjects who are at a high-risk for TB. The second goal was to compare the prevalence of these biomarkers in asymptomatic, CD4+ T cell-matched HIV+TB− subjects from India who are at high-risk for TB with similar subjects from US who are at low-risk for TB.

Methods and Results

Anti-MS and -MPT51 antibodies were assessed in sera from 480 subjects including PPD+ or PPD− healthy subjects, healthy community members, and HIV−TB+ and HIV+TB+ patients from India. Results demonstrate high sensitivity (∼80%) of detection of smear-positive HIV−TB+ and HIV+TB+ patients, and high specificity (>97%) with PPD+ subjects and endemic controls. While ∼45% of the asymptomatic HIV+TB− patients at high-risk for TB tested biomarker-positive, >97% of the HIV+TB− subjects at low risk for TB tested negative. Although the current studies are hampered by lack of knowledge of the outcome, these results provide strong support for the potential of these biomarkers to detect incipient, subclinical TB in HIV+ subjects.

Conclusions

These biomarkers provide high sensitivity and specificity for TB diagnosis in a TB endemic setting. Their performance is not compromised by concurrent HIV infection, site of TB and absence of pulmonary manifestations in HIV+TB+ patients. Results also demonstrate the potential of these biomarkers for identifying incipient subclinical TB in HIV+TB− subjects at high-risk for TB.

Cultures of Mycobacterium tuberculosis grown under oxygen depletion conditions enter into a state of nonreplicating persistence that may reflect a physiologically latent state. When these cultures were harvested and injected intranasally into mice, no bacteria could be recovered from the lungs for about 3 weeks, but after that evidence of regrowth was observed. Preimmunization of mice with a panel of selected vaccine candidates slowed or prevented this event. This simple model has potential for identifying vaccines targeting latent tuberculosis.

Although the global prevalence of leprosy has decreased over the last few decades due to an effective multidrug regimen, large numbers of new cases are still being reported, raising questions as to the ability to identify patients likely to spread disease and the effects of chemotherapy on the overall incidence of leprosy. This can partially be attributed to the lack of diagnostic markers for different clinical states of the disease and the consequent implementation of differential, optimal drug therapeutic strategies. Accordingly, comparative bioinformatics and Mycobacterium leprae protein microarrays were applied to investigate whether leprosy patients with different clinical forms of the disease can be categorized based on differential humoral immune response patterns. Evaluation of sera from 20 clinically diagnosed leprosy patients using native protein and recombinant protein microarrays revealed unique disease-specific, humoral reactivity patterns. Statistical analysis of the serological patterns yielded distinct groups that correlated with phenolic glycolipid I reactivity and clinical diagnosis, thus demonstrating that leprosy patients, including those diagnosed with the paucibacillary, tuberculoid form of disease, can be classified based on humoral reactivity to a subset of M. leprae protein antigens produced in recombinant form.

The 81-kDa malate synthase (MS; Rv 1837c) and the 27-kDa MPT51 (Rv 3803c) of Mycobacterium tuberculosis are immunodominant antigens recognized by serum antibodies from ∼80% of human immunodeficiency virus-negative smear-positive tuberculosis patients from India. We now provide evidence that the use of the MS/MPT51-based serodiagnostic assay can serve as an adjunct to sputum microscopy in the rapid diagnosis of pulmonary tuberculosis.

Background

Maternal schistosomiasis and filariasis have been shown to influence infant responses to neonatal bacille Calmette-Guérin (BCG) immunisation but the effects of maternal hookworm, and of de-worming in pregnancy, are unknown.

Methods

In Entebbe, Uganda, we conducted a randomised, double-blind, placebo-controlled trial of a single dose of 400 mg of albendazole in the second trimester of pregnancy. Neonates received BCG. Interferon-gamma (IFN-γ) and interleukin (IL)-5 responses to a mycobacterial antigen (crude culture filtrate proteins (CFP) of Mycobacterium tuberculosis) were measured in a whole blood assay. We analysed results for binary variables using χ2 tests and logistic regression. We analysed continuous variables using Wilcoxon's tests.

Results

Maternal hookworm was associated with reduced maternal IFN-γ responses to CFP (adjusted odds ratio for IFN-γ > median response: 0.14 (95% confidence interval 0.02–0.83, p = 0.021). Conversely, maternal hookworm was associated with subsequent increased IFN-γ responses in their one-year-old infants (adjusted OR 17.65 (1.20–258.66; p = 0.013)). Maternal albendazole tended to reduce these effects.

Conclusion

Untreated hookworm infection in pregnancy was associated with reduced maternal IFN-γ responses to mycobacterial antigens, but increased responses in their infants one year after BCG immunisation. The mechanisms of these effects, and their implications for protective immunity remain, to be determined.

Tuberculous meningitis (TBM) is the most common form of chronic infection of the central nervous system. Despite the magnitude of the problem, the general diagnostic outlook is discouraging. Specifically, there is no generally accepted early confirmative diagnosis protocol available for TBM. Various Mycobacterium tuberculosis antigens are now recognized as potential markers for diagnosis of TBM. However, their presence remains questionable, and many of these antigens are reported in the blood but not in the cerebrospinal fluid (CSF). This study identifies a specific protein marker in CSF which will be useful in early diagnosis of TBM. We have demonstrated the presence of a 30-kDa protein band in CSF of 100% (n = 5) of confirmed and 90% (n = 138) of suspected TBM patients out of 153 TBM patients. The 30-kDa band was excised from the gel, destained extensively, and digested with trypsin. The resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Partially purified proteins from CSF samples of TBM were analyzed by two-dimensional polyacrylamide gel electrophoresis and Western blotting. Immunoblotting and enzyme-linked immunosorbent assay (ELISA) were performed to confirm the presence of proteins in the 30-kDa protein band. The antigen 85 (Ag 85) complex was detected in CSF of TBM patients by indirect ELISA using antibodies against Ag 85 complex. The results of this study showed the 30-kDa protein band contained MTB proteins Rv3804c (Ag85A) and Rv1886c (Ag 85B), both members of the Ag85 complex. This was also confirmed by using immunotechniques such as indirect ELISA and the dot immunobinding assay. Detection of Ag85 complex was observed in CSF of 89% (71 out of 80) of suspected TBM patients that were 30-kDa protein positive. The observed 30-kDa protein in the CSF is comprised of the MTB Ag85 complex. This protein was earlier reported to be present in the blood of patients with extra-central nervous system tuberculosis. Therefore, this finding suggests that this protein can be used as a molecular marker for any type of tuberculous infection. It also provides a more sensitive immunoassay option for the early and confirmatory diagnosis of TBM.

A total of 104 polypeptides were purified from the low-molecular-mass secretory proteome of Mycobacterium tuberculosis H37Rv using a combination of anion exchange column chromatography and high resolution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution. The goal of this study was to identify polypeptides from a low-molecular-mass secretory proteome recognized by human subjects infected with M. tuberculosis and to ascertain the differences in specificity of antigen recognition by the peripheral blood mononuclear cells (PBMCs) and pleural fluid mononuclear cells (PFMCs) of these individuals. The study identified CFP-8 (Rv0496), CFP-11 (Rv2433c), CFP-14.5 (Rv2445c), and CFP-31 (Rv0831c) as novel T-cell antigens apart from previously characterized ESAT-6, TB10.4, CFP10, GroES, MTSP14, MTSP17, CFP21, MPT64, Ag85A, and Ag85B on the basis of recognition by PBMCs of tuberculosis contacts and treated tuberculosis patients. Further, polypeptides prominently recognized by PFMCs of tuberculous pleurisy patients were the same as those recognized by PBMCs of healthy contacts and treated tuberculosis patients. The results of our study indicate the homogeneity of antigenic target recognition by lymphocytes at the site of infection and at the periphery in the human subjects studied and the need to evaluate these antigenic targets as components of future antituberculous vaccines.

Structural analysis of compounds identified as lipid I and II from Mycobacterium smegmatis demonstrated that the lipid moiety is decaprenyl phosphate; thus, M. smegmatis is the first bacterium reported to utilize a prenyl phosphate other than undecaprenyl phosphate as the lipid carrier involved in peptidoglycan synthesis. In addition, mass spectrometry showed that the muropeptides from lipid I are predominantly N-acetylmuramyl-l-alanine-d-glutamate-meso-diaminopimelic acid-d-alanyl-d-alanine, whereas those isolated from lipid II form an unexpectedly complex mixture in which the muramyl residue and the pentapeptide are modified singly and in combination. The muramyl residue is present as N-acetylmuramic acid, N-glycolylmuramic acid, and muramic acid. The carboxylic functions of the peptide side-chains of lipid II showed three types of modification, with the dominant one being amidation. The preferred site for amidation is the free carboxyl group of the meso-diaminopimelic acid residue. Diamidated species were also observed. The carboxylic function of the terminal d-alanine of some molecules is methylated, as are all three carboxylic acid functions of other molecules. This study represents the first structural analysis of mycobacterial lipid I and II and the first report of extensive modifications of these molecules. The observation that lipid I was unmodified strongly suggests that the lipid II intermediates of M. smegmatis are substrates for a variety of enzymes that introduce modifications to the sugar and amino acid residues prior to the synthesis of peptidoglycan.

Trends in increased tuberculosis infection and a fatality rate of ∼23% have necessitated the search for alternative biomarkers using newly developed postgenomic approaches. Here we provide a systematic analysis of Mycobacterium tuberculosis (Mtb) by directly profiling its gene products. This analysis combines high-throughput proteomics and computational approaches to elucidate the globally expressed complements of the three subcellular compartments (the cell wall, membrane, and cytosol) of Mtb. We report the identifications of 1044 proteins and their corresponding localizations in these compartments. Genome-based computational and metabolic pathways analyses were performed and integrated with proteomics data to reconstruct response networks. From the reconstructed response networks for fatty acid degradation and lipid biosynthesis pathways in Mtb, we identified proteins whose involvements in these pathways were not previously suspected. Furthermore, the subcellular localizations of these expressed proteins provide interesting insights into the compartmentalization of these pathways, which appear to traverse from cell wall to cytoplasm. Results of this large-scale subcellular proteome profile of Mtb have confirmed and validated the computational network hypothesis that functionally related proteins work together in larger organizational structures.

Serum samples obtained from human immunodeficiency virus (HIV)-infected tuberculosis (TB) patients months prior to clinical TB were used to delineate the profile of Mycobacterium tuberculosis culture filtrate proteins recognized during subclinical TB. A subset of ∼12 antigens was recognized by antibodies in these serum samples. Antibodies to two of these antigens (81 [88]-kDa malate synthase [GlcB] and MPT51) were present in serum samples obtained during incipient subclinical TB in 19 (∼90%) of the 21 HIV-infected TB patients tested. These antigens will be useful for devising diagnostic tests that can identify HIV-positive individuals who are at a high risk for developing clinical TB.

Background

Mycobacterium avium are ubiquitous environmental organisms and a cause of disseminated infection in patients with end-stage AIDS. The glycopeptidolipids (GPL) of M. avium are proposed to participate in the pathogenesis of this organism, however, establishment of a clear role for GPL in disease production has been limited by the inability to genetically manipulate M. avium.

Methods

To be able to study the role of the GPL in M. avium pathogenesis, a ts-sacB selection system, not previously used in M. avium, was employed as a means to achieve homologous recombination for the rhamnosyltransferase (rtfA) gene of a pathogenic serovar 8 strain of M. avium to prevent addition of serovar-specific sugars to rhamnose of the fatty acyl-peptide backbone of GPL. The genotype of the resultant rtfA mutant was confirmed by polymerase chain reaction and southern hybridization. Disruption in the proximal sugar of the haptenic oligosaccharide resulted in the loss of serovar specific GPL with no change in the pattern of non-serovar specific GPL moieties as shown by thin layer chromatography and gas chromatography/mass spectrometry. Complementation of wild type (wt) rtfA in trans through an integrative plasmid restored serovar-8 specific GPL expression identical to wt serovar 8 parent strain.

Results

In this study, we affirm our results that rtfA encodes an enzyme responsible for the transfer of Rha to 6d-Tal and provide evidence of a second allelic exchange mutagenesis system suitable for M. avium.

Conclusion

We report the second allelic exchange system for M. avium utilizing ts-sacB as double-negative and xylE as positive counter-selection markers, respectively. This system of allelic exchange would be especially useful for M. avium strains that demonstrate significant isoniazid (INH) resistance despite transformation with katG. Through the construction of mutants in GPL or other mycobacterial components, their roles in M. avium pathogenesis, biosynthesis, or drug resistance can be studied in a consistent manner.