Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection.

Two mycobacteriophages were administered intravenously to a male with treatment-refractory Mycobacterium abscessus pulmonary infection and severe cystic fibrosis lung disease. The phages were engineered to enhance their capacity to lyse M. abscessus and were selected specifically as the most effective against the subject's bacterial isolate. In the setting of compassionate use, the evidence of phage-induced lysis was observed using molecular and metabolic assays combined with clinical assessments. M. abscessus isolates pre and post-phage treatment demonstrated genetic stability, with a general decline in diversity and no increased resistance to phage or antibiotics. The anti-phage neutralizing antibody titers to one phage increased with time but did not prevent clinical improvement throughout the course of treatment. The subject received lung transplantation on day 379, and systematic culturing of the explanted lung did not detect M. abscessus. This study describes the course and associated markers of a successful phage treatment of M. abscessus in advanced lung disease.

Structural implications of lipoarabinomannan glycans from global clinical isolates in diagnosis of Mycobacterium tuberculosis infection.

In Mycobacterium tuberculosis (Mtb), surface-exposed Lipoarabinomannan (LAM) is a key determinant of immunogenicity, yet its intrinsic heterogeneity confounds typical structure-function analysis. Recently, LAM gained a strong foothold as a validated marker for active tuberculosis (TB) infection and has shown great potential in new diagnostic efforts. However, no efforts have yet been made to model or evaluate the impact of mixed polyclonal Mtb infections (infection with multiple strains) on TB diagnostic procedures other than antibiotic susceptibility testing. Here, we selected three TB clinical isolates (HN878, EAI, and IO) and purified LAM from these strains to present an integrated analytical approach of one-dimensional and two-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy, as well as enzymatic digestion and site-specific mass spectrometry (MS) to probe LAM structure and behavior at multiple levels. Overall, we found that the glycan was similar in all LAM preparations, albeit with subtle variations. Succinates, lactates, hydroxybutyrate, acetate, and the hallmark of Mtb LAM-methylthioxylose (MTX), adorned the nonreducing terminal arabinan of these LAM species. Newly identified acetoxy/hydroxybutyrate was present only in LAM from EAI and IO Mtb strains. Notably, detailed LC/MS-MS unambiguously showed that all acyl modifications and the lactyl ether in LAM are at the 3-OH position of the 2-linked arabinofuranose adjacent to the terminal ß-arabinofuranose. Finally, after sequential enzymatic deglycosylation of LAM, the residual glycan that has ∼50% of α-arabinofuranose -(1→5) linked did not bind to monoclonal antibody CS35. These data clearly indicate the importance of the arabinan termini arrangements for the antigenicity of LAM.

Characterization of the Antigenic Heterogeneity of Lipoarabinomannan, the Major Surface Glycolipid of Mycobacterium tuberculosis, and Complexity of Antibody Specificities toward This Antigen.

Lipoarabinomannan (LAM), the major antigenic glycolipid of Mycobacterium tuberculosis, is an important immunodiagnostic target for detecting tuberculosis (TB) infection in HIV-1-coinfected patients, and is believed to mediate a number of functions that promote infection and disease development. To probe the human humoral response against LAM during TB infection, several novel LAM-specific human mAbs were molecularly cloned from memory B cells isolated from infected patients and grown in vitro. The fine epitope specificities of these Abs, along with those of a panel of previously described murine and phage-derived LAM-specific mAbs, were mapped using binding assays against LAM Ags from several mycobacterial species and a panel of synthetic glycans and glycoconjugates that represented diverse carbohydrate structures present in LAM. Multiple reactivity patterns were seen that differed in their specificity for LAM from different species, as well as in their dependence on arabinofuranoside branching and nature of capping at the nonreducing termini. Competition studies with mAbs and soluble glycans further defined these epitope specificities and guided the design of highly sensitive immunodetection assays capable of detecting LAM in urine of TB patients, even in the absence of HIV-1 coinfection. These results highlighted the complexity of the antigenic structure of LAM and the diversity of the natural Ab response against this target. The information and novel reagents described in this study will allow further optimization of diagnostic assays for LAM and may facilitate the development of potential immunotherapeutic approaches to inhibit the functional activities of specific structural motifs in LAM.

Structural determinants in a glucose-containing lipopolysaccharide from Mycobacterium tuberculosis critical for inducing a subset of protective T cells.

Mycobacteria synthesize intracellular, 6-O-methylglucose-containing lipopolysaccharides (mGLPs) proposed to modulate bacterial fatty acid metabolism. Recently, it has been shown that Mycobacterium tuberculosis mGLP specifically induces a specific subset of protective γ9δ2 T cells. Mild base treatment, which removes all the base-labile groups, reduces the specific activity of mGLP required for induction of these T cells, suggesting that acylation of the saccharide moieties is required for γ9δ2 T-cell activation. On the basis of this premise, we used analytical LC/MS and NMR methods to identify and locate the acyl functions on the mGLP saccharides. We found that mGLP is heterogeneous with respect to acyl functions and contains acetyl, isobutyryl, succinyl, and octanoyl groups and that all acylations in mGLP, except for succinyl and octanoyl residues, reside on the glucosyl residues immediately following the terminal 3-O-methylglucose. Our analyses also indicated that the octanoyl residue resides at position 2 of an internal glucose toward the reducing end. LC/MS analysis of the residual product obtained by digesting the mGLP with pancreatic α-amylase revealed that the product is an oligosaccharide terminated by α-(1→4)-linked 6-O-methyl-d-glucosyl residues. This oligosaccharide retained none of the acyl groups, except for the octanoyl group, and was unable to induce protective γ9δ2 T cells. This observation confirmed that mGLP induces γ9δ2 T cells and indicated that the acylated glucosyl residues at the nonreducing terminus of mGLP are required for this activity.

A Subset of Protective γ9δ2 T Cells Is Activated by Novel Mycobacterial Glycolipid Components.

γ9δ2 T cells provide a natural bridge between innate and adaptive immunity, rapidly and potently respond to pathogen infection in mucosal tissues, and are prominently induced by both tuberculosis (TB) infection and bacillus Calmette Guérin (BCG) vaccination. Mycobacterium-expanded γ9δ2 T cells represent only a subset of the phosphoantigen {isopentenyl pyrophosphate [IPP] and (E)-4-hydroxy-3-methyl-but-2-enylpyrophosphate [HMBPP]}-responsive γ9δ2 T cells, expressing an oligoclonal set of T cell receptor (TCR) sequences which more efficiently recognize and inhibit intracellular Mycobacterium tuberculosis infection. Based on this premise, we have been searching for M. tuberculosis antigens specifically capable of inducing a unique subset of mycobacterium-protective γ9δ2 T cells. Our screening strategy includes the identification of M. tuberculosis fractions that expand γ9δ2 T cells with biological functions capable of inhibiting intracellular mycobacterial replication. Chemical treatments of M. tuberculosis whole-cell lysates (MtbWL) ruled out protein, nucleic acid, and nonpolar lipids as the M. tuberculosis antigens inducing protective γ9δ2 T cells. Mild acid hydrolysis, which transforms complex carbohydrate to monomeric residues, abrogated the specific activity of M. tuberculosis whole-cell lysates, suggesting that a polysaccharide was required for biological activity. Extraction of MtbWL with chloroform-methanol-water (10:10:3) resulted in a polar lipid fraction with highly enriched specific activity; this activity was further enriched by silica gel chromatography. A combination of mass spectrometry and nuclear magnetic resonance analysis of bioactive fractions indicated that 6-O-methylglucose-containing lipopolysaccharides (mGLP) are predominant components present in this active fraction. These results have important implications for the development of new immunotherapeutic approaches for prevention and treatment of TB.

Importance of specimen pretreatment for the low-level detection of mycobacterial lipoarabinomannan in human serum.

Patient care and prevention of disease outbreaks rely heavily on the performance of diagnostic tests. These tests are typically carried out in serum, urine, and other complex sample matrices, but are often plagued by a number of matrix effects such as nonspecific adsorption and complexation with circulating proteins. This paper demonstrates the importance of sample pretreatment to overcome matrix effects, enabling the low-level detection of a disease marker for tuberculosis (TB). The impact of pretreatment is illustrated by detecting a cell wall component unique to mycobacteria, lipoarabinomannan (LAM). LAM is a major virulence factor in the infectious pathology of Mycobacterium tuberculosis (Mtb) and has been successfully detected in the body fluids of TB-infected individuals; however, its clinical sensitivity - identifying patients with active infection - remains problematic. This and the companion paper show that the detection of LAM in an immunoassay is plagued by its complexation with proteins and other components in serum. Herein, we present the procedures and results from an investigation of several different pretreatment schemes designed to disrupt complexation and thereby improve detection. These sample pretreatment studies, aimed at determining the optimal conditions for complex disruption, were carried out by using a LAM simulant derived from the nonpathogenic M. smegmatis, a mycobacterium often used as a model for Mtb. We have found that a perchloric acid-based pretreatment step improves the ability to detect this simulant by ∼1500× with respect to that in untreated serum. This paper describes the approach to pretreatment, how pretreatment improves the detection of the LAM simulant in human serum, and the results from a preliminary investigation to identify possible contributors to complexation by fractionating serum according to molecular weight. The companion paper applies this pretreatment approach to assays of TB patient samples.

Detection of the tuberculosis antigenic marker mannose-capped lipoarabinomannan in pretreated serum by surface-enhanced Raman scattering.

The ability to detect tuberculosis (TB) continues to be a global health care priority. This paper describes the development and preliminary assessment of the clinical accuracy of a heterogeneous immunoassay that integrates a serum pretreatment process with readout by surface-enhanced Raman scattering (SERS) for the low-level detection of mannose-capped lipoarabinomannan (ManLAM). ManLAM is a major virulence factor in the infectious pathology of Mycobacterium tuberculosis (Mtb) that has been found in the serum and other body fluids of infected patients. The effectiveness of ManLAM as a TB diagnostic marker, however, remains unproven for reasons not yet well understood. As reported herein, we have found that (1) ManLAM complexes with proteins and possibly other components in serum; (2) these complexes have a strongly detrimental impact on the ability to detect ManLAM using an immunoassay; (3) a simple pretreatment step can disrupt this complexation; and (4) disruption by pretreatment improves detection by 250×. We also describe the results from a preliminary assessment on the utility of serum pretreatment by running immunoassays on archived specimens from 24 TB-positive patients and 10 healthy controls. ManLAM was measurable in 21 of the 24 TB-positive specimens, but not in any of the 10 control specimens. These findings, albeit for a very small specimen set, translate to a clinical sensitivity of 87.5% and a clinical specificity of 100%. Together, these results both provide much needed evidence for the clinical utility of ManLAM as a TB marker, and demonstrate the potential utility of our overall approach to serve as a new strategy for the development of diagnostic tests for this disease.

A single arabinan chain is attached to the phosphatidylinositol mannosyl core of the major immunomodulatory mycobacterial cell envelope glycoconjugate, lipoarabinomannan.

Lipoarabinomannan (LAM) is composed of a phosphatidylinositol anchor followed by a mannan followed by an arabinan that may be capped with various motifs including oligosaccharides of mannose. A related polymer, lipomannan (LM), is composed of only the phosphatidylinositol and mannan core. Both the structure and the biosynthesis of LAM have been studied extensively. However, fundamental questions about the branching structure of LM and the number of arabinan chains on the mannan backbone in LAM remain. LM and LAM molecules produced by three different glycosyltransferase mutants of Mycobacterium smegmatis were used here to investigate these questions. Using an MSMEG_4241 mutant that lacks the α-(1,6)-mannosyltransferase used late in LM elongation, we showed that the reducing end region of the mannan that is attached to inositol has 5-7 unbranched α-6-linked-mannosyl residues followed by two or three α-6-linked mannosyl residues branched with single α-mannopyranose residues at O-2. After these branched mannosyl residues, the α-6-linked mannan chain is terminated with an α-mannopyranose at O-2 rather than O-6 of the penultimate residue. Analysis of the number of arabinans attached to the mannan core of LM in two other mutants (ΔembC and ΔMSMEG_4247) demonstrated exactly one arabinosyl substitution of the mannan core suggestive of the arabinosylation of a linear LM precursor with ∼10-12 mannosyl residues followed by additional mannosylation of the core and arabinosylation of a single arabinosyl "primer." Thus, these studies suggest that only a single arabinan chain attached near the middle of the mannan core is present in mature LAM and allow for an updated working model of the biosynthetic pathway of LAM and LM.

Tuberculosis in the African continent: A comprehensive review.

Tuberculosis continues to be a major global health problem, causing an estimated 8.8 million new cases and 1.45 million deaths annually. New drugs in the 1940s made it possible to beat the disease, and consequently, the number of cases reduced drastically. Fast-forward a few decades, drugresistant strains of varied virulence are reported consistently, disease is again on the rise and the treatment has not kept pace. Tuberculosis is the leading cause of death among HIV-infected persons in many resource-constrained settings however, it is curable and preventable. The unprecedented growth of the tuberculosis epidemic in Africa is attributable to several factors, the most important being the HIV epidemic. Analysis of molecular-based data have shown diverse genetic backgrounds among both drug-sensitive and MDR TB isolates in Africa presumably due to underlying genetic and environmental differences. The good news is that there have been important advances recently in TB drugs and diagnostics. Despite the availability of revolutionary tests that allow for faster diagnosis and of new drugs and regimens that offer better and safer treatment it is now becoming clearer that national efforts on TB control should be enhanced and focus on improving the quality of prevention, diagnosis, treatment and care services; strengthening program management, implementation and supervision. This review is an assessment of the trend in TB in Africa.

Prospective Analysis of urINe LAM to Eliminate NTM Sputum Screening (PAINLESS) study: Rationale and trial design for testing urine lipoarabinomannan as a marker of NTM lung infection in cystic fibrosis.

Background: Routine screening for nontuberculous mycobacterial (NTM) lung disease is dependent on sputum cultures. This is particularly challenging in the cystic fibrosis (CF) population due to reduced sputum production and low culture sensitivity. Biomarkers of infection that do not rely on sputum may lead to earlier diagnosis, but validation trials require a unique prospective design. Purpose: The rationale of this trial is to investigate the utility of urine lipoarabinomannan (LAM) as a test to identify people with CF with a new positive NTM culture. We hypothesize that urine LAM is a sensitive, non-invasive screening test with a high negative predictive value to identify individuals with a relatively low risk of having positive NTM sputum culture. Study design: This is a prospective, single-center, non-randomized observational study in adults with CF, 3 years of negative NTM cultures, and no known history of NTM positive cultures. Patients are followed for two year-long observational periods with the primary endpoint being a positive NTM sputum culture within a year of a positive urine LAM result and a secondary endpoint of a positive NTM sputum culture within 3 years of a positive urine LAM result. Study implementation includes remote consent and sample collection to accommodate changes from the COVID-19 pandemic. Conclusions: This report describes the study design of an observational study aimed at using a urine biomarker to assist in the diagnosis of NTM lung infection in pwCF. If successful, urine LAM could be used as an adjunct to traditional sputum cultures for routine NTM screening.

Correction: Performance of novel antibodies for lipoarabinomannan to develop diagnostic tests for Mycobacterium tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0274415.].

Collected Thoughts on Mycobacterial Lipoarabinomannan, a Cell Envelope Lipoglycan.

The presence of lipoarabinomannan (LAM) in the Mycobacterium tuberculosis (Mtb) cell envelope was first reported close to 100 years ago. Since then, numerous studies have been dedicated to the isolation, purification, structural definition, and elucidation of the biological properties of Mtb LAM. In this review, we present a brief historical perspective on the discovery of Mtb LAM and the herculean efforts devoted to structurally characterizing the molecule because of its unique structural and biological features. The significance of LAM remains high to this date, mainly due to its distinct immunological properties in conjunction with its role as a biomarker for diagnostic tests due to its identification in urine, and thus can serve as a point-of-care diagnostic test for tuberculosis (TB). In recent decades, LAM has been thoroughly studied and massive amounts of information on this intriguing molecule are now available. In this review, we give the readers a historical perspective and an update on the current knowledge of LAM with information on the inherent carbohydrate composition, which is unique due to the often puzzling sugar residues that are specifically found on LAM. We then guide the readers through the complex and myriad immunological outcomes, which are strictly dependent on LAM's chemical structure. Furthermore, we present issues that remain unresolved and represent the immediate future of LAM research. Addressing the chemistry, functions, and roles of LAM will lead to innovative ways to manipulate the processes that involve this controversial and fascinating biomolecule.

Monoclonal antibodies to lipoarabinomannan/arabinomannan - characteristics and implications for tuberculosis research and diagnostics.

Antibodies to the mycobacterial surface lipoglycan lipoarabinomannan (LAM) and its related capsular polysaccharide arabinomannan (AM) are increasingly important for investigations focused on both understanding mechanisms of protection against Mycobacterium tuberculosis (Mtb) and developing next-generation point-of-care tuberculosis (TB) diagnostics. We provide here an overview of the growing pipeline of monoclonal antibodies (mAbs) to LAM/AM. Old and new methodologies for their generation are reviewed and we outline and discuss their glycan epitope specificity and other features with implications for the TB field.

Culture independent markers of nontuberculous mycobacterial (NTM) lung infection and disease in the cystic fibrosis airway.

Nontuberculous mycobacteria (NTM) are opportunistic pathogens that affect a relatively small but significant portion of the people with cystic fibrosis (CF), and may cause increased morbidity and mortality in this population. Cultures from the airway are the only test currently in clinical use for detecting NTM. Culture techniques used in clinical laboratories are insensitive and poorly suited for population screening or to follow progression of disease or treatment response. The lack of sensitive and quantitative markers of NTM in the airway impedes patient care and clinical trial design, and has limited our understanding of patterns of acquisition, latency and pathogenesis of disease. Culture-independent markers of NTM infection have the potential to overcome many of the limitations of standard NTM cultures, especially the very slow growth, inability to quantitate bacterial burden, and low sensitivity due to required decontamination procedures. A range of markers have been identified in sputum, saliva, breath, blood, urine, as well as radiographic studies. Proposed markers to detect presence of NTM or transition to NTM disease include bacterial cell wall products and DNA, as well as markers of host immune response such as immunoglobulins and the gene expression of circulating leukocytes. In all cases the sensitivity of culture-independent markers is greater than standard cultures; however, most do not discriminate between various NTM species. Thus, each marker may be best suited for a specific clinical application, or combined with other markers and traditional cultures to improve diagnosis and monitoring of treatment response.

Metabolomic signatures in guinea pigs infected with epidemic-associated W-Beijing strains of Mycobacterium tuberculosis.

With the understanding that the laboratory propagated strain of Mycobacterium tuberculosis H37Rv is of modest virulence and is drug susceptible, in the present study, we performed a nuclear magnetic resonance-based metabolomic analysis of lung tissues and serum obtained from guinea pigs infected by low dose aerosol exposure to clinical isolates of Mycobacterium tuberculosis. High Resolution Magic Angle Spinning NMR coupled with multivariate statistical analysis of 159 lung tissues obtained from multiple locations of age-matched naïve and 30 and 60 days of infected guinea pig lungs revealed a wide dispersal of metabolic patterns, but within these, distinct clusters of signatures could be seen that differentiated between naive control and infected animals. Several metabolites were identified that changed in concert with the progression of each infection. Major metabolites that could be interpreted as indicating host glutaminolysis were consistent with activated host immune cells encountering increasingly hypoxic conditions in the necrotic lung lesions. Moreover, glutathione levels were constantly elevated, probably in response to oxygen radical production in these lesions. Additional distinct signatures were also seen in infected serum, with altered levels of several metabolites. Multivariate statistical analysis clearly differentiated the infected from the uninfected sera; in addition, Receiver Operator Characteristic curve generated with principal component 1 scores showed an area under the curve of 0.908. These data raise optimism that discrete metabolomic signatures can be defined that can predict the progression of the tuberculosis disease process, and form the basis of an innovative and rapid diagnostic process.

Isolation of a distinct Mycobacterium tuberculosis mannose-capped lipoarabinomannan isoform responsible for recognition by CD1b-restricted T cells.

Mannose-capped lipoarabinomannan (ManLAM) is a complex lipoglycan abundantly present in the Mycobacterium tuberculosis cell envelope. Many biological properties have been ascribed to ManLAM, from directly interacting with the host and participating in the intracellular survival of M. tuberculosis, to triggering innate and adaptive immune responses, including the activation of CD1b-restricted T cells. Due to its structural complexity, ManLAM is considered a heterogeneous population of molecules which may explain its different biological properties. The presence of various modifications such as fatty acids, succinates, lactates, phosphoinositides and methylthioxylose in ManLAM have proven to correlate directly with its biological activity and may potentially be involved in the interactions between CD1b and the T cell population. To further delineate the specific ManLAM epitopes involved in CD1b-restricted T cell recognition, and their potential roles in mediating immune responses in M. tuberculosis infection, we established a method to resolve ManLAM into eight different isoforms based on their different isoelectric values. Our results show that a ManLAM isoform with an isoelectric value of 5.8 was the most potent in stimulating the production of interferon-γ in different CD1b-restricted T-cell lines. Compositional analyses of these isoforms of ManLAM revealed a direct relationship between the overall charge of the ManLAM molecule and its capacity to be presented to T cells via the CD1 compartment.

The mycobacterial acyltransferase PapA5 is required for biosynthesis of cell wall-associated phenolic glycolipids.

Phenolic glycolipids (PGLs) are non-covalently bound components of the outer membrane of many clinically relevant mycobacterial pathogens, and play important roles in pathogen biology. We report a mutational analysis that conclusively demonstrates that the conserved acyltransferase-encoding gene papA5 is essential for PGL production. In addition, we provide an in vitro acyltransferase activity analysis that establishes proof of principle for the competency of PapA5 to utilize diol-containing polyketide compounds of mycobacterial origin as acyl-acceptor substrates. Overall, the results reported herein are in line with a model in which PapA5 catalyses the acylation of diol-containing polyketides to form PGLs. These studies advance our understanding of the biosynthesis of an important group of mycobacterial glycolipids and suggest that PapA5 might be an attractive target for exploring the development of antivirulence drugs.

Production of mycobacterial cell wall glycopeptidolipids requires a member of the MbtH-like protein family.

BACKGROUND: Glycopeptidolipids (GPLs) are among the major free glycolipid components of the outer membrane of several saprophytic and clinically-relevant Mycobacterium species. The architecture of GPLs is based on a constant tripeptide-amino alcohol core of nonribosomal peptide synthetase origin that is N-acylated with a 3-hydroxy/methoxy acyl chain synthesized by a polyketide synthase and further decorated with variable glycosylation patterns built from methylated and acetylated sugars. GPLs have been implicated in many aspects of mycobacterial biology, thus highlighting the significance of gaining an understanding of their biosynthesis. Our bioinformatics analysis revealed that every GPL biosynthetic gene cluster known to date contains a gene (referred herein to as gplH) encoding a member of the MbtH-like protein family. Herein, we sought to conclusively establish whether gplH was required for GPL production. RESULTS: Deletion of gplH, a gene clustered with nonribosomal peptide synthetase-encoding genes in the GPL biosynthetic gene cluster of Mycobacterium smegmatis, produced a GPL deficient mutant. Transformation of this mutant with a plasmid expressing gplH restored GPL production. Complementation was also achieved by plasmid-based constitutive expression of mbtH, a paralog of gplH found in the biosynthetic gene cluster for production of the siderophore mycobactin of M. smegmatis. Further characterization of the gplH mutant indicated that it also displayed atypical colony morphology, lack of sliding motility, altered capacity for biofilm formation, and increased drug susceptibility. CONCLUSIONS: Herein, we provide evidence formally establishing that gplH is essential for GPL production in M. smegmatis. Inactivation of gplH also leads to a pleiotropic phenotype likely to arise from alterations in the cell envelope due to the lack of GPLs. While genes encoding MbtH-like proteins have been shown to be needed for production of siderophores and antibiotics, our study presents the first case of one such gene proven to be required for production of a cell wall component. Furthermore, our results provide the first example of a mbtH-like gene with confirmed functional role in a member of the Mycobacterium genus. Altogether, our findings demonstrate a critical role of gplH in mycobacterial biology and advance our understanding of the genetic requirements for the biosynthesis of an important group of constituents of the mycobacterial outer membrane.

A bioanalytical method to determine the cell wall composition of Mycobacterium tuberculosis grown in vivo.

Mycobacterium tuberculosis bacilli exhibit cell wall alterations during in vivo growth. Development of ultrasensitive analytical techniques with high specificities is required to analyze the cell wall of M. tuberculosis isolated from experimental animals because of the low amounts of bacteria available and contamination by host tissue. Here we present a novel methodology to analyze all three major components (mycolic acids, arabinogalactan, and peptidoglycan) of the mycobacterial cell wall from mycobacteria isolated from animal tissue. In this procedure, the cell wall carbohydrates are analyzed by gas chromatography tandem mass spectrometry (GC/MS/MS) of alditol acetates, the peptidoglycan by GC/MS (mass spectrometry) analysis of the unique amino acid diaminopimelic acid (after derivatization with isopropyl chloroformate), and the mycolic acids by liquid chromatography (LC)/MS (negative ion) without derivatization. The procedure was designed so that all three analyses could be performed starting with a single sample given the difficulty of preparing multiple aliquots in known ratios. Linkage analysis, including an enantiomeric specific procedure, of the arabinogalactan polymer is also presented. These procedures will enable the determination of the cell wall alterations known to occur in the important nongrowing "dormant" M. tuberculosis present during disease. With some adaptations, the methodology is also applicable to the analysis of small amounts of in vivo grown bacteria of other species.

Identification of serological biomarkers of infection, disease progression and treatment efficacy for leprosy.

Although leprosy is curable with drug treatment, the identification of biomarkers of infection, disease progression and treatment efficacy would greatly help to reduce the overall prevalence of the disease. Reliable biomarkers would also reduce the incidence of grade-2 disability by ensuring that those who are most at risk are diagnosed and treated early or offered repeated treatments in the case of relapse. In this study, we examined the reactivity of sera from lepromatous and tuberculoid leprosy patients (LPs) against a panel of 12 recombinant Mycobacterium leprae proteins and found that six proteins were strongly recognised by multibacillary (MB) patients, while only three were consistently recognised by paucibacillary patients. To better understand the dynamics of patient antibody responses during and after drug therapy, we measured antibody titres to four recombinant proteins, phenolic glycolipid-I and lipoarabinomannan at baseline and up to two years after diagnosis to investigate the temporal changes in the antibody titres. Reactivity patterns to individual antigens and decreases in antibody titres were patient-specific. Antibody titres to proteins declined more rapidly vs. those to carbohydrate and glycolipid antigens. Compared to baseline values, increases in antibody titres were observed during reactional episodes in one individual. Additionally, antibody responses against a subset of antigens that provided a good prognostic indicator of disease progression were analysed in 51 household contacts of MB index cases for up to two years. Although the majority of these contacts showed no change or exhibited decreases in antibody titres, seven individuals developed higher titres towards one or more of these antigens and one individual with progressively higher titres was diagnosed with borderline lepromatous leprosy 19 months after enrolment. The results of this study indicate that antibody titres to specific M. leprae antigens can be used to monitor treatment efficacy in LPs and assess disease progression in those most at risk for developing this disease.