Characteristic profile of antibody responses to PPD, ESAT-6, and CFP-10 of Mycobacterium tuberculosis in pulmonary tuberculosis suspected cases in Surabaya, Indonesia

Accurate and rapid diagnostic tools are important aspects of managing tuberculosis (TB) cases appropriately. However, the sensitivity and specificity of diagnostic kits based on immune response such as the tuberculin skin test (TST) and interferon gamma release assay (IGRA) are still debated. Thus, the exploration and assessment of specific biomarker-targeted antibodies are needed for the development of an accurate and rapid diagnostic tool. The present study was conducted in patients with a respiratory problem suspected to be TB at Dr. Soetomo Hospital, Surabaya, Indonesia. Among 102 patients tested by GeneXpert and AFB, 59 serum samples were from cases retrospectively determined to have active TB. A total of 102 serum of healthy controls (HC) was also collected. The PPD antigen and the recombinant CFP-10 and ESAT-6 proteins were prepared. Antibody responses against these proteins were evaluated by ELISA. All samples were also screened for the possibility of Mycobacterium avium-intracellulare complex (MAC) infection using Capilla MaC kit. The results showed that TB patients had a significantly higher concentration of IgG antibody in response to PPD than the HC. In addition, the receiver operating characteristic (ROC) curve analysis showed that PPD was acceptable for diagnostic purposes with an AUC value of 0.835 (95% CI 0.770-0.900, p < 0.0001). However, ESAT-6 and CFP-10 had low AUCs, and 32 samples from both groups showed a low concentration of IgA antibody against all antigens. The MAC detection results also showed that the concentration of IgA in the HC group was the highest. The current results indicate that PPD is a better antigen for antibody-based detection of TB than ESAT-6 and CFP-10. Based on the MAC detection assay, 53 people in the HC group were probably infected with rapidly growing nontuberculous mycobacteria (NTM), although antibody response to PPD was low

Proteome Analysis Identifies the Dpr Protein of Streptococcus mutans as an Important Factor in the Presence of Early Streptococcal Colonizers of Tooth Surfaces

Oral streptococci are primary colonizers of tooth surfaces and Streptococcus mutans is the principal causative agent of dental caries in humans. A number of proteins are involved in the formation of monospecies biofilms by S. mutans. This study analyzed the protein expression profiles of S. mutans biofilms formed in the presence or absence of S. gordonii, a pioneer colonizer of the tooth surface, by two-dimensional gel electrophoresis (2-DE). After identifying S. mutans proteins by Mass spectrometric analysis, their expression in the presence of S. gordonii was analyzed. S. mutans was inoculated with or without S. gordonii DL1. The two species were compartmentalized using 0.2-μl Anopore membranes. The biofilms on polystyrene plates were harvested, and the solubilized proteins were separated by 2-DE. When S. mutans biofilms were formed in the presence of S. gordonii, the peroxide resistance protein Dpr of the former showed 4.3-fold increased expression compared to biofilms that developed in the absence of the pioneer colonizer. In addition, we performed a competition assay using S. mutans antioxidant protein mutants together with S. gordonii and other initial colonizers. Growth of the dpr-knockout S. mutans mutant was significantly inhibited by S. gordonii, as well as by S. sanguinis. Furthermore, a cell viability assay revealed that the viability of the dpr-defective mutant was significantly attenuated compared to the wild-type strain when co-cultured with S. gordonii. Therefore, these results suggest that Dpr might be one of the essential proteins for S. mutans survival on teeth in the presence of early colonizing oral streptococci

A Histone-Like Protein of Mycobacteria Possesses Ferritin Superfamily Protein-Like Activity and Protects against DNA Damage by Fenton Reaction

Iron is an essential metal for living organisms but its level must be strictly controlled in cells, because ferrous ion induces toxicity by generating highly active reactive oxygen, hydroxyl radicals, through the Fenton reaction. In addition, ferric ion shows low solubility under physiological conditions. To overcome these obstacles living organisms possess Ferritin superfamily proteins that are distributed in all three domains of life: bacteria, archaea, and eukaryotes. These proteins minimize hydroxyl radical formation by ferroxidase activity that converts Fe2+ into Fe3+ and sequesters iron by storing it as a mineral inside a protein cage. In this study, we discovered that mycobacterial DNA-binding protein 1 (MDP1), a histone-like protein, has similar activity to ferritin superfamily proteins. MDP1 prevented the Fenton reaction and protects DNA by the ferroxidase activity. The Km values of the ferroxidase activity by MDP1 of Mycobacterium bovis bacillus Calmette-Guérin (BCG-3007c), Mycobacterium tuberculosis (Rv2986c), and Mycobacterium leprae (ML1683; ML-LBP) were 0.292, 0.252, and 0.129 mM, respectively. Furthermore, one MDP1 molecule directly captured 81.4±19.1 iron atoms, suggesting the role of this protein in iron storage. This study describes for the first time a ferroxidase-iron storage protein outside of the ferritin superfamily proteins and the protective role of this bacterial protein from DNA damage

Serological Surveillance Development for Tropical Infectious Diseases Using Simultaneous Microsphere-Based Multiplex Assays and Finite Mixture Models

Background:A strategy to combat infectious diseases, including neglected tropical diseases (NTDs), will depend on the development of reliable epidemiological surveillance methods. To establish a simple and practical seroprevalence detection system, we developed a microsphere-based multiplex immunoassay system and evaluated utility using samples obtained in Kenya.Methods:We developed a microsphere-based immuno-assay system to simultaneously measure the individual levels of plasma antibody (IgG) against 8 antigens derived from 6 pathogens: Entamoeba histolytica (C-IgL), Leishmania donovani (KRP42), Toxoplasma gondii (SAG1), Wuchereria bancrofti (SXP1), HIV (gag, gp120 and gp41), and Vibrio cholerae (cholera toxin). The assay system was validated using appropriate control samples. The assay system was applied for 3411 blood samples collected from the general population randomly selected from two health and demographic surveillance system (HDSS) cohorts in the coastal and western regions of Kenya. The immunoassay values distribution for each antigen was mathematically defined by a finite mixture model, and cut-off values were optimized.Findings:Sensitivities and specificities for each antigen ranged between 71 and 100%. Seroprevalences for each pathogen from the Kwale and Mbita HDSS sites (respectively) were as follows: HIV, 3.0% and 20.1%; L. donovani, 12.6% and 17.3%; E. histolytica, 12.8% and 16.6%; and T. gondii, 30.9% and 28.2%. Seroprevalences of W. bancrofti and V. cholerae showed relatively high figures, especially among children. The results might be affected by immunological cross reactions between W. bancrofti-SXP1 and other parasitic infections; and cholera toxin and the enterotoxigenic E. coli (ETEC), respectively.Interpretation:A microsphere-based multi-serological assay system can provide an opportunity to comprehensively grasp epidemiological features for NTDs. By adding pathogens and antigens of interest, optimized made-to-order high-quality programs can be established to utilize limited resources to effectively control NTDs in Africa

Nutritional status positively impacts humoral immunity against its Mycobacterium tuberculosis, disease progression, and vaccine development.

Nutritional status contributes to the regulation of immune responses against pathogens, and malnutrition has been considered as a risk factor for tuberculosis (TB). Mycobacterium tuberculosis (Mtb), the causative agent of TB, can modulate host lipid metabolism and induce lipid accumulation in macrophages, where the bacilli adopt a dormant phenotype. In addition, serum lipid components play dual roles in the regulation of and protection from Mtb infection. We analyzed the relationship between nutritional status and the humoral immune response in TB patients. We found that serum HDL levels are positively correlated with the serum IgA specific for Mtb antigens. Analysis of the relationship between serum nutritional parameters and clinical parameters in TB patients showed that serum albumin and CRP levels were negatively correlated before treatment. We also observed reduced serum LDL levels in TB patients following treatment. These findings may provide insight into the role of serum lipids in host immune responses against Mtb infection. Furthermore, improving the nutritional status may enhance vaccination efficacy

<i>S</i>. <i>mutans</i> Dpr expression by Western blotting analysis.

<p>(A) Planktonic cells. (B) Biofilm. Lane M, molecular mass markers; lane 1, <i>S</i>. <i>mutans</i> without <i>S</i>. <i>gordonii</i>; lane 2, <i>S</i>. <i>mutans</i> co-cultured with <i>S</i>. <i>gordonii</i> DL1; lane 3, <i>S</i>. <i>mutans</i> co-cultured with <i>S</i>. <i>gordonii</i> Δ<i>spxB</i>.</p

Viability assay of <i>S</i>. <i>mutans</i> strains after co-inoculation with <i>S</i>. <i>gordonii</i>.

<p><i>S</i>. <i>mutans</i> strains were inoculated into <i>S</i>. <i>gordonii</i> pre-existing BHI medium and additionally inoculated at 37°C under anaerobic conditions for 48 h. <i>S</i>. <i>mutans</i> CFU on MS agar plates supplemented with bacitracin were counted and adjusted to CFU/well. Data are shown as the means of triplicate platings from one of two reproducible experiments. *<i>P</i> < 0.05.</p

Primers used in this study.

<p>^<i>a</i>Endonuclease restriction sites are underlined.</p><p>^<i>b</i>Restriction sites were not included.</p><p>Primers used in this study.</p

Inhibition of the growth of <i>S</i>. <i>mutans</i> strains by <i>S</i>. <i>gordonii</i>.

<p>(A) Inhibition of the growth of <i>S</i>. <i>mutans dpr</i>-deficient strains by <i>S</i>. <i>gordonii</i> DL1 and <i>spxB</i>-deficient strain. <i>S</i>. <i>gordonii</i> strains were inoculated first and grown for 24 h at 37°C in an aerobic atmosphere. Then, <i>S</i>. <i>mutans</i> strains were inoculated next to these colonizers, and the plates were incubated for 24 h (upper lane). <i>S</i>. <i>gordonii</i> and <i>S</i>. <i>mutans</i> were inoculated simultaneously on the plate and incubated for 24 h at 37°C under aerobic conditions (lower lane). (B) Inhibition of the growth of <i>S</i>. <i>mutans sod</i>-, <i>ahpC</i>-, <i>dpr</i>- <i>ahpC</i>, and <i>sod</i> double mutants by <i>S</i>. <i>gordonii</i> DL1. The culture conditions were the same as in (A).</p

The strains and plasmids used in this study.

<p>^<i>a</i> Em^s, erythromycin-sensitive.</p><p>^<i>b</i> Em^r, erythromycin-resistant.</p><p>^<i>c</i> Spc^r, spectinomycin-resistant.</p><p>^<i>d</i> Km^r, kanamycin-resistant.</p><p>^<i>e</i> Amp^r, ampicillin-resistant</p><p>^<i>f</i>. KDU, Culture collection of the Division of Community Oral Health Science, Kyushu Dental University, Kitakyushu, Japan.</p><p>^<i>g</i> RIKEN, Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Japan.</p><p>The strains and plasmids used in this study.</p