A Review Of Host-Specific Diagnostic And Surrogate Biomarkers In Children With Pulmonary Tuberculosis.

BACKGROUND: Tuberculosis (TB) is one of the most common causes of mortality globally with a steady rise in paediatric cases in the past decade. Laboratory methods of diagnosing TB and monitoring response to treatment have limitations. Current research focuses on interrogating host- and/or pathogen-specific biomarkers to address this problem. METHODS: We reviewed the literature on host-specific biomarkers in TB to determine their value in diagnosis and treatment response in TB infected and HIV/TB co-infected children on anti-tuberculosis treatment. RESULTS AND CONCLUSION: While no single host-specific biomarker has been identified for diagnosis or treatment responses in children, several studies suggest predictive biosignatures for disease activity. Alarmingly, current data on host-specific biomarkers for diagnosing and assessing anti-tuberculosis treatment in TB/HIV co-infected children is inadequate. Various factors affecting host-specific biomarker responses should be considered in interpreting findings and designing future studies within specific clinical settings.

A computational method for the prediction and functional analysis of potential Mycobacterium tuberculosis adhesin-related proteins.

OBJECTIVES: Mycobacterial adherence plays a major role in the establishment of infection within the host. Adhesin-related proteins attach to host receptors and cell-surface components. The current study aimed to utilize in-silico strategies to determine the adhesin potential of conserved hypothetical (CH) proteins. METHODS: Computational analysis was performed on the whole Mycobacterium tuberculosis H37Rv proteome using a software program for the prediction of adhesin and adhesin-like proteins using neural networks (SPAAN) to determine the adhesin potential of CH proteins. A robust pipeline of computational analysis tools: Phyre2 and pFam for homology prediction; Mycosub, PsortB, and Loctree3 for subcellular localization; SignalP-5.0 and SecretomeP-2.0 for secretory prediction, were utilized to identify adhesin candidates. RESULTS: SPAAN revealed 776 potential adhesins within the whole MTB H37Rv proteome. Comprehensive analysis of the literature was cross-tabulated with SPAAN to verify the adhesin prediction potential of known adhesin (n = 34). However, approximately a third of known adhesins were below the probability of adhesin (Pad) threshold (Pad ≥0.51). Subsequently, 167 CH proteins of interest were categorized using essential in-silico tools. CONCLUSION: The use of SPAAN with supporting in-silico tools should be fundamental when identifying novel adhesins. This study provides a pipeline to identify CH proteins as functional adhesin molecules.

M. tuberculosis curli pili (MTP) facilitates a reduction of microbicidal activity of infected THP-1 macrophages during early stages of infection.

Mycobacterium tuberculosis (M. tuberculosis) curli pili (MTP) is a surface located adhesin, which is involved in the initial point-of-contact between the pathogen and the host. Host-pathogen interaction is essential for establishing infection. M. tuberculosis has the ability to infect various host lung cell types, which includes both the epithelial cells and macrophages, and subsequent differences in their cellular function will be evident in their metabolic profiles. Understanding the differences between these cell types and their individual metabolic response to M. tuberculosis infection, with and without the presence of the MTP, will aid to better elucidate the role of this adhesin in modulating metabolic pathways during infection. This may further contribute to the development of improved diagnostic and therapeutic interventions, much needed at present in order to improve control the global tuberculosis (TB) epidemic. This study used a two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC-TOFMS) metabolomics approach to compare the metabolite profiles of A549 epithelial cells to that of THP-1 macrophages, infected with M. tuberculosis, in the presence and absence of MTP. Significant metabolites were identified using various univariate and multivariate statistical analysis. A total of 44, 40, 50 and 34 metabolites were differentially detected when comparing the (a) uninfected A549 epithelial cells to uninfected THP-1 macrophages, (b) wild-type infected A549 epithelial cells to wild-type infected THP-1 macrophages, (c) ∆mtp-infected A549 epithelial cells to ∆mtp-infected THP-1 macrophages (d) complement-infected A549 epithelial cells to complement-infected THP-1 macrophages, respectively. These included metabolites that were involved in amino acid metabolism, fatty acid metabolism, general central carbon metabolism, and nucleic acid metabolism. In the absence of the M. tuberculosis MTP adhesin, the THP-1 macrophages predominantly displayed higher concentrations of amino acids and their metabolic intermediates, than the A549 epithelial cells. The deletion of MTP from M. tuberculosis in the host infection models potentially elicited a pro-inflammatory phenotype, particularly in the macrophage model. In the presence of MTP, the metabolite profile changes indicate potential regulation of host defence mechanisms, accompanied by a reduction in microbicidal abilities of host cells. Hence MTP can be considered a virulence factor of M. tuberculosis. Therefore, blocking MTP interaction with the host may facilitate a faster pathogen clearance during the initial stages of infection, and potentially enhance current therapeutic interventions.

Predominance of the Mycobacterium tuberculosis Beijing strain amongst children from a high tuberculosis burden township in South Africa.

The objective was to determine the molecular epidemiology and drug susceptibility patterns of Mycobacterium tuberculosis (MTB) of children and their household contacts (HHC) in Umlazi, a high TB-burden township in South Africa. Sixty eight MTBRifPLUS positive TB-infected children (TIC) (≤14 years) and 111 HHC were enrolled. Drug susceptibility testing (DST) was performed on sputum samples using the proportion method and GenoType® MTBDR. Genotyping of MTB was conducted using IS6110-restriction fragment length polymorphism (RFLP) and spoligotyping. Rifampicin (RIF) susceptibility was observed in 67/68 TIC. GenoType® MTBDRplus and phenotypic DST identified drug resistant strains in five of 16 culture-confirmed TIC. The Beijing strain was identified in six and the F15/LAM4/KZN strain in one of the 13 TIC respectively. Four patients with unknown RFLP strains belonged to spoligoclades S, T1, T3 variant and X2. The S-lineage and an unknown strain were identified in two HHC. MDR-TB and pre-XDR-TB were identified in one HHC each. Household transmission could not be determined as none of the culture-confirmed TIC resided with the six culture-confirmed contacts. The predominance of the hypervirulent Beijing strain and presence of drug-resistant strains must be considered in the implementation of effective TB control strategies and development of efficacious vaccines.

Genome sequence of a carbapenemase-encoding Acinetobacter baumannii isolate of the sequence type 231 isolated from hospital wastewater in South Africa.

OBJECTIVES: The resistome, virulome, mobilome and phylogenetic relationship of the Acinetobacter baumannii isolate FG121 depicting the multilocus sequence type (ST) 231 isolated from hospital effluent water in South Africa was determined using whole-genome sequence analysis. METHOD: A. baumannii FG121 was isolated on Leed Acinetobacter Medium (LAM) agar and the bacterial isolate was identified using the VITEK®2 platform. Antibiotic susceptibility testing was performed using Kirby-Bauer Disk diffusion method. A whole genome sequencing library was constructed from DNA extracted from the isolate using the Illumina Nextera XT library preparation kit and was sequenced using the Illumina NextSeq500 platform. Generated reads were de novo assembled using SpAdes v.3.9. The assembled contigs were annotated, and multilocus sequence type, antimicrobial resistance, and virulence genes were identified. RESULTS: The resistome was consistent with the resistance phenotype of the isolate with resistance determinants for beta-lactams, aminoglycosides, and tetracycline (blaADC-25, blaOXA-23, blaOXA-51, blaNDM-1, aph[3']-VIa and tet[B]). Global phylogenomic analysis using BacWGSTdb revealed that the isolate belonged to the multilocus sequence type ST-231, similar to previously reported isolates from South Africa, the United States, and related to the invasive KR3831 isolate identified from Oman in 2012, suggesting the isolate might be imported from abroad. Virulome analysis predicted both virulence and biofilm-determinants of A. baumannii, which may help to establish infections in adverse conditions. CONCLUSION: This is the first report on a carbapenemase-encoding A. baumannii ST-231 isolated from hospital effluent water. Our data will offer insight into the global phylogenetic, pathogenicity and distribution of A. baumannii in South Africa.

Antibiotic resistance and biofilm formation of Acinetobacter baumannii isolated from high-risk effluent water in tertiary hospitals in South Africa.

OBJECTIVES: Discharge of drug-resistant, biofilm-forming pathogens from hospital effluent water into municipal wastewater treatment plants poses a public health concern. This study examined the relationship between antibiotic resistance levels and biofilm formation of Acinetobacter baumannii strains isolated from hospital effluents. METHODS: Antibiotic susceptibility of 71 A. baumannii isolates was evaluated by the Kirby-Bauer disk diffusion method. Minimum inhibitory concentrations (MICs) were determined by the agar dilution method, while the minimum biofilm eradication concentration (MBEC) was determined by the broth dilution method. Genotyping was performed for plasmid DNA. Biofilm formation was evaluated by the microtitre plate method and was quantified using crystal violet. A P-value of <0.05 was regarded as statistically significant in all tests. RESULTS: Extensively drug-resistant (XDR) strains made up 58% of the isolates, while multidrug-resistant (MDR) and pandrug-resistant (PDR) strains made up 50% of the isolates from final effluent. The MBEC of ciprofloxacin increased by 255-fold, while that of ceftazidime was as high as 63-1310-fold compared with their respective MICs. Isolates were classified into four plasmid pattern groups with no association between biofilm formation and plasmid type (P = 0.0921). The degree of biofilm formation was independent of the level of antibiotic resistance, although MDR, XDR and PDR isolates produced significant biofilm biomass (P = 0.2580). CONCLUSION: These results suggest that hospital effluent is a potential source of MDR biofilm-forming A. baumannii strains. Appropriate treatment and disposal of effluents are essential to prevent the presence of drug-resistant pathogens in wastewater.

M. tuberculosis curli pili (MTP) is associated with alterations in carbon, fatty acid and amino acid metabolism in a THP-1 macrophage infection model.

The initial host-pathogen interaction is crucial for the establishment of infection. An improved understanding of the pathophysiology of Mycobacterium tuberculosis (M. tuberculosis) during macrophage infection can aid the development of intervention therapeutics against tuberculosis. M. tuberculosis curli pili (MTP) is a surface located adhesin, involved in the first point-of-contact between pathogen and host. This study aimed to better understand the role of MTP in modulating the intertwined metabolic pathways of M. tuberculosis and its THP-1 macrophage host. Metabolites were extracted from pelleted wet cell mass of THP-1 macrophages infected with M. tuberculosis wild-type V9124 (WT), Δmtp-deletion mutant and the mtp-complemented strains, respectively, via a whole metabolome extraction method using a 1:3:1 ratio of chloroform:methanol:water. Metabolites were detected by two-dimensional gas chromatography time-of-flight mass spectrometry. Significant metabolites were determined through univariate and multivariate statistical tests and online pathway databases. Relative to the WT, a total of nine and ten metabolites were significantly different in the Δmtp and complement strains, respectively. All nine significant metabolites were found in elevated levels in the Δmtp relative to the WT. Additionally, of the ten significant metabolites, eight were detected in lower levels and two were detected in higher levels in the complement relative to the WT. The absence of the MTP adhesin resulted in reduced virulence of M. tuberculosis leading to alterations in metabolites involved in carbon, fatty acid and amino acid metabolism during macrophage infection, suggesting that MTP plays an important role in the modulation of host metabolic activity. These findings support the prominent role of the MTP adhesin as a virulence factor as well as a promising biomarker for possible diagnostic and therapeutic intervention.

Light Forge: A Microfluidic DNA Melting-based Tuberculosis Test.

BACKGROUND: There is a well-documented lack of rapid, low-cost tuberculosis (TB) drug resistance diagnostics in low-income settings across the globe. It is these areas that are plagued with a disproportionately high disease burden and in greatest need of these diagnostics. METHODS: In this study, we compared the performance of Light Forge, a microfluidic high-resolution melting analysis (HRMA) prototype for rapid low-cost detection of TB drug resistance with a commercial HRMA device, a predictive "nearest-neighbor" thermodynamic model, DNA sequencing, and phenotypic drug susceptibility testing (DST). The initial development and assessment of the Light Forge assay was performed with 7 phenotypically drug resistant strains of Mycobacterium tuberculosis (M.tb) that had their rpoB gene subsequently sequenced to confirm resistance to Rifampin. These isolates of M.tb were then compared against a drug-susceptible standard, H37Rv. Seven strains of M.tb were isolated from clinical specimens and individually analyzed to characterize the unique melting profile of each strain. RESULTS: Light Forge was able to detect drug-resistance linked mutations with 100% concordance to the sequencing, phenotypic DST and the "nearest neighbor" thermodynamic model. Researchers were then blinded to the resistance profile of the seven M.tb strains. In this experiment, Light Forge correctly classified 7 out of 9 strains as either drug resistant or drug susceptible. CONCLUSIONS: Light Forge represents a promising prototype for a fast, low-cost diagnostic alternative for detection of drug resistant strains of TB in resource constrained settings.

Immunoscreening of the M. tuberculosis F15/LAM4/KZN secretome library against TB patients' sera identifies unique active- and latent-TB specific biomarkers.

Tuberculosis (TB) protein biomarkers are urgently needed for the development of point-of-care diagnostics, new drugs and vaccines. Mycobacterium tuberculosis extracellular and secreted proteins play an important role in host-pathogen interactions. Antibodies produced against M. tuberculosis proteins before the onset of clinical symptoms can be used in proteomic studies to identify their target proteins. In this study, M. tuberculosis F15/LAM4/KZN strain phage secretome library was screened against immobilized polyclonal sera from active TB patients (n = 20), TST positive individuals (n = 15) and M. tuberculosis uninfected individuals (n = 20) to select and identify proteins recognized by patients' antibodies. DNA sequence analysis from randomly selected latent TB and active TB specific phage clones revealed 118 and 96 ORFs, respectively. Proteins essential for growth, virulence and metabolic pathways were identified using different TB databases. The identified active TB specific biomarkers included five proteins, namely, TrpG, Alr, TreY, BfrA and EspR, with no human homologs, whilst latent TB specific biomarkers included NarG, PonA1, PonA2 and HspR. Future studies will assess potential applications of identified protein biomarkers as TB drug or vaccine candidates/targets and diagnostic markers with the ability to discriminate LTBI from active TB.

Evaluation of a synthetic peptide for the detection of anti-Mycobacterium tuberculosis curli pili IgG antibodies in patients with pulmonary tuberculosis.

Tuberculosis (TB) remains a serious threat in underdeveloped areas. Mycobacterium tuberculosis curli pili (MTP), a virulence factor, is a potential biomarker for a reliable point of care (POC) test and was evaluated for its ability to react with Immunoglobulin G (IgG) in TB patients. An MTP synthetic peptide in a slot blot assay was used to screen serum/plasma samples (n = 65) in 3 separate cohorts, including 40 TB positive (16 HIV co-infected), 20 TB negative/HIV negative patients and 5 healthy volunteers. Forty samples were true positives (HIV positive, n = 16), 23 true negatives (HIV negative) and 2 false positives (HIV negative). The McNemar test demonstrated a 3.08% accuracy estimate (CI: -2.1% - 3.08%). This confirms that MTP is expressed during infection, including HIV-TB co-infection, is likely to be suitable for the design of a POC test and supports the validation of MTP for TB detection in larger patient populations.

Mycobacterium tuberculosis strains induce strain-specific cytokine and chemokine response in pulmonary epithelial cells.

M. tuberculosis F15/LAM4/KZN has been associated with high transmission rates of drug resistant tuberculosis in the KwaZulu-Natal province of South Africa. The current study elucidated the cytokine/chemokine responses induced by representatives of the F15/LAM4/KZN and other dominant strain families in pulmonary epithelial cells. Multiplex cytokine analyses were performed at 24, 48 and 72h post infection of the A549 pulmonary epithelial cell line with the F15/LAM4/KZN, F28, F11, Beijing, Unique and H37Rv strains at an MOI of ∼10:1. Twenty-three anti- and pro-inflammatory cytokines/chemokines were detected at all-time intervals. Significantly high concentrations of IL-6, IFN-γ, TNF-α and G-CSF at 48h, and IL-8, IFN-γ, TNF-α, G-CSF and GM-CSF at 72h, were induced by the F28 and F15/LAM4/KZN strains, respectively. Lower levels of cytokines/chemokines were induced by either the Beijing or Unique strains at all three time intervals. All strains induced up-regulation of pathogen recognition receptors (PRRs) (TLR3 and TLR5) while only the F15/LAM4/KZN, F11 and F28 strains induced significant differential expression of TLR2 compared to the Beijing, Unique and H37Rv strains. The low induction of cytokines in epithelial cells by the Beijing strain correlates with its previously reported hypervirulent properties. High concentrations of cytokines and chemokines required for early protection against M. tuberculosis infections induced by the F15/LAM4/KZN and F28 strains suggests a lower virulence of these genotypes compared to the Beijing strain. These findings demonstrate the high diversity in host cytokine/chemokine response to early infection of pulmonary epithelial cells by different strains of M. tuberculosis.

Fitness-compensatory mutations facilitate the spread of drug-resistant F15/LAM4/KZN and F28 Mycobacterium tuberculosis strains in KwaZulu-Natal, South Africa.

While the acquisition of drug resistance is often accompanied by fitness costs, Mycobacterium tuberculosis has developed mechanisms to overcome these costs in the form of compensatory mutations. In an attempt to dissect strain-specific differences in biological fitness, 10 M. tuberculosis genomes, representing F15/LAM4/KZN, Beijing, F11 and F28 genotypes were sequenced on the Illumina MiSeq platform. Drug-susceptible F15/LAM4/KZN strains differed by 43 SNPs, demonstrating that heterogeneity exists even among closely-related strains. We found unique, nonsynonymous single-nucleotide polymorphisms (SNPs) in the sigA and grcC1 genes of multidrug resistant (MDR) and XDR F15/LAM4/KZN strains, respectively. The F28 MDR strain harboured a novel ubiA mutation in combination with its embB M306I mutation, which may be related to ethambutol resistance. In addition, it possessed a low-frequency rpoC mutation, suggesting that this strain was in the process of developing compensation. In contrast, no compensatory mutations were identified in Beijing and F11 MDR strains, corroborating its low in vitro fitness. Clinical strains also harboured unique SNPs in a number of important genes associated with virulence, highlighting the need for future studies which examine the correlation of genetic variations with phenotypic diversity. In summary, whole-genome sequencing revealed the presence of fitness-compensatory mutations in F15/LAM4/KZN and F28 genotypes which predominate in MDR and/or extensively drug resistant (XDR) forms in KwaZulu-Natal, South Africa.

Analysis of clinical and microbiological data on Acinetobacter baumannii strains assist the preauthorization of antibiotics at the patient level for an effective antibiotic stewardship program.

Drug resistant Acinetobacter baumannii (A. baumannii) poses serious treatment challenges and is on the rise worldwide. The Infectious Diseases Society of America/Society for Healthcare Epidemiology of America recommends preauthorization of antibiotics to ensure successful antibiotic stewardship programs (ASWPs). This study estimates and analyzes the microbiological and clinical characteristics of A. baumanii strains with differentiating criteria for sepsis versus colonization, in order to support preauthorization and assist ASWPs at the patient level. A retrospective observational study was performed from 2008 to 2014. The clinical and microbiological characteristics of A. baumannii strains were correlated to assess pathogenic status and antibiotic resistance patterns. A flow chart was produced to differentiate between sepsis and colonization amongst patient groups. A. baumannii was cultured in 2656 cases, with a prevalence of 0.9-2.4% during 7 years study periods. There was a statistically significant difference between the sepsis and colonization groups (P=0.02). Sepsis accounted for 37-51% of A. baumanii isolates and colonisation for 49-63% (P=<0.01). Multidrug resistant (MDR), extensive drug resistant (XDR) and pandrug resistant (PDR) A. baumannii was detected in 53-60%, 1-19% and 1% of cultures in the sepsis group, and 75%, 8-23% and 1% in the colonized group. There was a high percentage of polymicrobial infection in the sepsis group and pure growth was not always significant for sepsis. Cases of MDR and XDR A. baumannii increased over the seven-year study, while PDR strains emerged. For a successful ASWP, both clinical and microbiological information should be interpreted when establishing preauthorization/decision to treat.

Identification of unique essential proteins from a Mycobacterium tuberculosis F15/LAM4/KZN phage secretome library.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis disease (TB), the leading cause of death from bacterial infection worldwide. Although treatable, the resurgence of multidrug-resistant and extensively drug-resistant TB is a major setback for the fight against TB globally. Consequently, there is an urgent need for new Mtb-derived biomarkers for use in the design of new drugs and rapid point-of-care diagnostic or prognostic tools for the management of TB transmission. Therefore, the present study aimed to identify unique Mtb-secreted proteins from the extensively drug-resistant Mtb F15/LAM4/KZN phage secretome library. A whole genome library was constructed using genomic DNA fragments of the Mtb F15/LAM4/KZN strain. A phage secretome sub-library of 8 × 103 clones was prepared and phage DNA was sequenced from 120 randomly selected clones. DNA sequence BLAST analysis identified 86 open reading frames. Using bioinformatics tools and databases, 10 proteins essential for in vivo growth and survival of Mtb (Nrp, PssA, MmpL5, SirA, GatB, EspA, TopA, EccCa1, Rv1634 and Rv3103c) were identified. Proteins essential for the growth and survival of Mtb during infection have potential application in the development of diagnostic tools, new drugs and vaccines. Further studies will be conducted to evaluate their potential application in the fight against TB.

Bacterial pili, with emphasis on Mycobacterium tuberculosis curli pili: potential biomarkers for point-of care tests and therapeutics.

CONTEXT: Novel biomarkers are essential for developing rapid diagnostics and therapeutic interventions Objective: This review aimed to highlight biomarker characterisation and assessment of unique bacterial pili. METHODS: A PubMed search for bacterial pili, diagnostics, vaccine and therapeutics was performed, with emphasis on the well characterised pili. RESULTS: In total, 46 papers were identified and reviewed. CONCLUSION: Extensive analyses of pili enabled by advanced nanotechnology and whole genome sequencing provide evidence that they are strong biomarker candidates. Mycobacterium tuberculosis curli pili are emphasised as important epitopes for the development of much needed point-of-care diagnostics and therapeutics.

Identification of Mycobacterium tuberculosis adherence-mediating components: a review of key methods to confirm adhesin function.

Anti-adhesion therapy represents a potentially promising avenue for the treatment and prevention of tuberculosis in a post-antibiotic era. Adhesins are surface-exposed microbial structures or molecules that enable pathogenic organisms to adhere to host surfaces, a fundamental step towards host infection. Although several Mycobacterium tuberculosis adhesins have been identified, it is predicted that numerous additional adherence-mediating components contribute to the virulence and success of this pathogen. Significant further research to discern and characterize novel M. tuberculosis adhesins is, therefore, required to gain a holistic account of M. tuberculosis adhesion to the host. This would enable the identification of potential drug and vaccine targets for attenuating M. tuberculosis adherence and infectivity. Several methods have been successfully applied to the study and identification of M. tuberculosis adhesins. In this manuscript, we review these methods, which include adherence assays that utilize wild-type and gene knockout mutant strains, epitope masking and competitive inhibition analyses, extracellular matrix protein binding assays, microsphere adhesion assays, M. tuberculosis auto-aggregation assays, and in silico analyses.

Mycobacterium tuberculosis strains exhibit differential and strain-specific molecular signatures in pulmonary epithelial cells.

Although pulmonary epithelial cells are integral to innate and adaptive immune responses during Mycobacterium tuberculosis infection, global transcriptomic changes in these cells remain largely unknown. Changes in gene expression induced in pulmonary epithelial cells infected with M. tuberculosis F15/LAM4/KZN, F11, F28, Beijing and Unique genotypes were investigated by RNA sequencing (RNA-Seq). The Illumina HiSeq 2000 platform generated 50 bp reads that were mapped to the human genome (Hg19) using Tophat (2.0.10). Differential gene expression induced by the different strains in infected relative to the uninfected cells was quantified and compared using Cufflinks (2.1.0) and MeV (4.0.9), respectively. Gene expression varied among the strains with the total number of genes as follows: F15/LAM4/KZN (1187), Beijing (1252), F11 (1639), F28 (870), Unique (886) and H37Rv (1179). A subset of 292 genes was commonly induced by all strains, where 52 genes were down-regulated while 240 genes were up-regulated. Differentially expressed genes were compared among the strains and the number of induced strain-specific gene signatures were as follows: F15/LAM4/KZN (138), Beijing (52), F11 (255), F28 (55), Unique (186) and H37Rv (125). Strain-specific molecular gene signatures associated with functional pathways were observed only for the Unique and H37Rv strains while certain biological functions may be associated with other strain signatures. This study demonstrated that strains of M. tuberculosis induce differential gene expression and strain-specific molecular signatures in pulmonary epithelial cells. Specific signatures induced by clinical strains of M. tuberculosis can be further explored for novel host-associated biomarkers and adjunctive immunotherapies.

Evaluation of the role of Mycobacterium tuberculosis pili (MTP) as an adhesin, invasin, and cytokine inducer of epithelial cells

Abstract This study was undertaken in order to assess the involvement of Mycobacterium tuberculosis pili (MTP) as an adhesin, invasin, and cytokine inducer in the M. tuberculosis-epithelial cell interaction. A MTP-deficient strain of M. tuberculosis demonstrated a significant reduction of 69.39% (p = 0.047) and 56.20% (p = 0.033) in its ability to adhere to and invade A549 pulmonary epithelial cells, respectively, in comparison with the wild-type strain. Complementation of the MTP-deficient mutant restored its adhesion and invasion capacity back to the wild-type levels. Overall, it was found that similar concentrations of IL-1β, IL-4, IL-6, IL-8, G-CSF, IFN-γ, MCP-1, and TNF-α were induced in A549 cells infected with the MTP-proficient and MTP-deficient strains. However, at 48 h post-infection, the MTP-deficient mutant induced significantly lower levels of TNF-α than the wild-type strain (p = 0.033). Furthermore, at 72 h post-infection, the mutant induced significantly higher levels of IL-8 than the wild-type (p = 0.005). We conclude that MTP is an adhesin/invasin of epithelial cells and, while playing a role in M. tuberculosis entry, they do not appear to largely influence the epithelial cell cytokine response.

Canonical pathways, networks and transcriptional factor regulation by clinical strains of Mycobacterium tuberculosis in pulmonary alveolar epithelial cells.

Limited knowledge exists on pathways, networks and transcriptional factors regulated within epithelial cells by diverse Mycobacterium tuberculosis genotypes. This study aimed to elucidate these mechanisms induced in A549 epithelial cells by dominant clinical strains in KwaZulu-Natal, South Africa. RNA for sequencing was extracted from epithelial cells at 48 h post-infection with 5 strains at a multiplicity of infection of approximately 10:1. Bioinformatics analysis performed with the RNA-Seq Tuxedo pipeline identified differentially expressed genes. Changes in pathways, networks and transcriptional factors were identified using Ingenuity Pathway Analysis (IPA). The interferon signalling and hepatic fibrosis/hepatic stellate cell activation pathways were among the top 5 canonical pathways in all strains. Hierarchical clustering for enrichment of cholesterol biosynthesis and immune associated pathways revealed similar patterns for Beijing and Unique; F15/LAM4/KZN and F11; and, F28 and H37Rv strains, respectively. However, the induction of top scoring networks varied among the strains. Among the transcriptional factors, only EHL, IRF7, PML, STAT1, STAT2 and VDR were induced by all clinical strains. Activation of the different pathways, networks and transcriptional factors revealed in the current study may be an underlying mechanism that results in the differential host response by clinical strains of M. tuberculosis.