Hairong Huang

Proteome-wide lysine acetylation profiling of the human pathogen Mycobacterium tuberculosis

N(ɛ)-Acetylation of lysine residues represents a pivotal post-translational modification used by both eukaryotes and prokaryotes to modulate diverse biological processes. Mycobacterium tuberculosis is the causative agent of tuberculosis, one of the most formidable public health threats. Many aspects of the biology of M. tuberculosis remain elusive, in particular the extent and function of N(ɛ)-lysine acetylation. With a combination of anti-acetyllysine antibody-based immunoaffinity enrichment with high-resolution mass spectrometry, we identified 1128 acetylation sites on 658 acetylated M. tuberculosis proteins. GO analysis of the acetylome showed that acetylated proteins are involved in the regulation of diverse cellular processes including metabolism and protein synthesis. Six types of acetylated peptide sequence motif were revealed from the acetylome. Twenty lysine-acetylated proteins showed homology with acetylated proteins previously identified from Escherichia coli, Salmonella enterica, Bacillus subtilis and Streptomyces roseosporus, with several acetylation sites highly conserved among four or five bacteria, suggesting that acetylated proteins are more conserved. Notably, several proteins including isocitrate lyase involved in the persistence, virulence and antibiotic resistance are acetylated, and site-directed mutagenesis of isocitrate lyase acetylation site to glutamine led to a decrease of the enzyme activity, indicating major roles of KAc in these proteins engaged cellular processes. Our data firstly provides a global survey of M. tuberculosis acetylation, and implicates extensive regulatory role of acetylation in this pathogen. This may serve as an important basis to address the roles of lysine acetylation in M. tuberculosis metabolism, persistence and virulence.

Precision methylome characterization of Mycobacterium tuberculosis complex (MTBC) using PacBio single-molecule real-time (SMRT) technology

Tuberculosis (TB) remains one of the most common infectious diseases caused by Mycobacterium tuberculosis complex (MTBC). To panoramically analyze MTBCs genomic methylation, we completed the genomes of 12 MTBC strains (Mycobacterium bovis; M. bovis BCG; M. microti; M. africanum; M. tuberculosis H37Rv; H37Ra; and 6 M. tuberculosis clinical isolates) belonging to different lineages and characterized their methylomes using single-molecule real-time (SMRT) technology. We identified three m6A sequence motifs and their corresponding methyltransferase (MTase) genes, including the reported mamA, hsdM and a newly discovered mamB. We also experimentally verified the methylated motifs and functions of HsdM and MamB. Our analysis indicated the MTase activities varied between 12 strains due to mutations/deletions. Furthermore, through measuring ‘the methylated-motif-site ratio’ and ‘the methylated-read ratio’, we explored the methylation status of each modified site and sequence-read to obtain the ‘precision methylome’ of the MTBC strains, which enabled intricate analysis of MTase activity at whole-genome scale. Most unmodified sites overlapped with transcription-factor binding-regions, which might protect these sites from methylation. Overall, our findings show enormous potential for the SMRT platform to investigate the precise character of methylome, and significantly enhance our understanding of the function of DNA MTase.

Biochip System for Rapid and Accurate Identification of Mycobacterial Species from Isolates and Sputum

ABSTRACT The accurate detection of mycobacterial species from isolates and clinical samples is important for pathogenic diagnosis and treatment and for disease control. There is an urgent need for the development of a rapid, simple, and accurate detection method. We established a biochip assay system, including a biochip, sample preparation apparatus, hybridization instrument, chip washing machine, and laser confocal scanner equipped with interpretation software for automatic diagnosis. The biochip simultaneously identified 17 common mycobacterial species by targeting the differences in the 16S rRNA. The system was assessed with 64 reference strains and 296 Mycobacterium tuberculosis and 243 nontuberculous mycobacterial isolates, as well as 138 other bacteria and 195 sputum samples, and then compared to DNA sequencing. The entire biochip assay took 6 h. The concordance rate between the biochip assay and the DNA sequencing results was 100%. In conclusion, the biochip system provides a simple, rapid, reliable, and highly accurate clinical assay for determination of mycobacterial species in a 6-h procedure, from either culture isolates or sputum samples, allowing earlier pathogen-adapted antimicrobial therapy in patients.

The essential mycobacterial amidotransferase GatCAB is a modulator of specific translational fidelity.

Although regulation of translation fidelity is an essential process1–7, diverse organisms and organelles have differing requirements of translational accuracy8–15, and errors in gene translation serve an adaptive function under certain conditions16–20. Therefore, optimal levels of fidelity may vary according to context. Most bacteria utilize a two-step pathway for the specific synthesis of aminoacylated glutamine and/or asparagine tRNAs, involving the glutamine amidotransferase GatCAB21–25, but it had not been appreciated that GatCAB may play a role in modulating mistranslation rates. Here, by using a forward genetic screen, we show that the mycobacterial GatCAB enzyme complex mediates the translational fidelity of glutamine and asparagine codons. We identify mutations in gatA that cause partial loss of function in the holoenzyme, with a consequent increase in rates of mistranslation. By monitoring single-cell transcription dynamics, we demonstrate that reduced gatCAB expression leads to increased mistranslation rates, which result in enhanced rifampicin-specific phenotypic resistance. Consistent with this, strains with mutations in gatA from clinical isolates of Mycobacterium tuberculosis show increased mistranslation, with associated antibiotic tolerance, suggesting a role for mistranslation as an adaptive strategy in tuberculosis. Together, our findings demonstrate a potential role for the indirect tRNA aminoacylation pathway in regulating translational fidelity and adaptive mistranslation.

Species identification of Mycobacterium abscessus subsp. abscessus and Mycobacterium abscessus subsp. bolletii using rpoB and hsp65, and susceptibility testing to eight antibiotics.

OBJECTIVES To separate Mycobacterium abscessus subsp. bolletii from Mycobacterium abscessus subsp. abscessus using species identification, and to investigate the in vitro activity of amikacin, cefoxitin, imipenem, levofloxacin, moxifloxacin, clarithromycin, azithromycin, and linezolid against Mycobacterium abscessus. METHODS Seventy M. abscessus isolates, previously identified by 16S rRNA sequencing, were further identified by comparative sequence analysis of rpoB and hsp65. Drug susceptibility testing was conducted using the microplate Alamar Blue assay in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines and interpreted using CLSI breakpoints. RESULTS Of the 70 strains, 45 (64%) were M. abscessus subsp. abscessus and 25 (36%) were M. abscessus subsp. bolletii. The majority of M. abscessus isolates were susceptible to azithromycin, amikacin, linezolid, and imipenem (M. abscessus subsp. abscessus: 93%, 98%, 93%, and 73%, respectively; M. abscessus subsp. bolletii: 96%, 96%, 80%, and 68%, respectively). Approximately half of the M. abscessus isolates were moderately susceptible to cefoxitin and moxifloxacin (M. abscessus subsp. abscessus 53% and 49%; M. abscessus subsp. bolletii 72% and 68%). Nearly all the M. abscessus isolates were resistant to levofloxacin (M. abscessus subsp. abscessus 96%, M. abscessus subsp. bolletii 100%). Inducible clarithromycin resistance was found in M. abscessus. After 14 days of incubation, 83% M. abscessus subsp. abscessus and 36% M. abscessus subsp. bolletii were resistant to clarithromycin. CONCLUSIONS Using rpoB and hsp65, M. abscessus subsp. bolletii could be distinguished from M. abscessus subsp. abscessus. Amikacin and azithromycin showed excellent activity against M. abscessus in vitro. Imipenem, linezolid, cefoxitin, and moxifloxacin also showed good activity. Levofloxacin was inactive against M. abscessus. Although clarithromycin showed excellent activity against M. abscessus on day 3, inducible resistance occurred, and after 14 days clarithromycin showed little activity against M. abscessus subsp. abscessus, but still had good activity against M. abscessus subsp. bolletii.

Xpert MTB/RIF and GenoType MTBDRplus assays for the rapid diagnosis of bone and joint tuberculosis

BACKGROUND Bone and joint tuberculosis (BJTB) constitutes about 10-20% of the extrapulmonary tuberculosis (EPTB) cases in China. The GenoType MTBDRplus assay (MTBDR) has been endorsed by the World Health Organization (WHO) for the diagnosis of pulmonary TB (PTB), while the Xpert MTB/RIF assay (Xpert) has also been endorsed by the WHO for the diagnosis of both PTB and EPTB. The diagnostic utility of these two techniques for BJTB was investigated prospectively. METHODS Sixty pus specimens were obtained from orthopedic patients. Smear, culture, Xpert, and MTBDR assays were performed for each specimen, and MGIT 960-based drug susceptibility testing (DST) was conducted for all of the isolates recovered. The diagnostic efficiency of Xpert and MTBDR was evaluated on the basis of bacteriological examination and the composite reference standard (CRS). RESULTS Fifty of the 60 patients were considered to have BJTB according to the CRS. The sensitivities of smear, culture, Xpert, and MTBDR were 26% (13/50), 48% (24/50), 82% (41/50), and 72% (36/50) respectively, while the specificities of all of the tests were 100% (10/10). Xpert was 100% concordant with MGIT 960-based DST for the detection of rifampicin resistance. MTBDR had a sensitivity of 83.3% and a specificity of 100% for the detection of rifampicin resistance and a sensitivity of 85.7% and specificity of 100% for the detection of isoniazid resistance. CONCLUSION With their high sensitivities, short turnaround times, and ability to diagnose TB and detect drug resistance simultaneously, both Xpert and MTBDR are feasible as diagnostic tools for BJTB in clinical practice.

Genetic Determinants Involved in p-Aminosalicylic Acid Resistance in Clinical Isolates from Tuberculosis Patients in Northern China from 2006 to 2012

ABSTRACT p-Aminosalicylic acid (PAS) is an important compound for treating multidrug-resistant tuberculosis (TB). Previous studies showed that thyA mutations are often related to PAS resistance in clinical isolates. We performed a systematic analysis of isolate genotypes and detected mutations in three folate pathway genes (folC, thyA, and ribD) in 61.1% (127/208) of PAS-resistant isolates, including 11 double mutants. This result expands our knowledge about the distribution and frequency of mutations related to PAS resistance in mycobacterial clinical isolates.

Latently and uninfected healthcare workers exposed to TB make protective antibodies against Mycobacterium tuberculosis

Significance It is not known whether natural immunity to TB exists or whether antibodies play an important role in immunity against infection by Mycobacterium tuberculosis (Mtb), the causative agent of TB. Here, we identify that a significant minority of healthcare workers who are exposed to high doses of Mtb make protective antibodies against infection as measured in both mouse infection and in vitro models. Some of these individuals had no prior evidence of latent TB infection, suggesting that they may represent a subset of “restrictors” who can control infection by Mtb. These results have important implications in the rational development of both protective and therapeutic vaccines against TB. The role of Igs in natural protection against infection by Mycobacterium tuberculosis (Mtb), the causative agent of TB, is controversial. Although passive immunization with mAbs generated against mycobacterial antigens has shown protective efficacy in murine models of infection, studies in B cell-depleted animals only showed modest phenotypes. We do not know if humans make protective antibody responses. Here, we investigated whether healthcare workers in a Beijing TB hospital—who, although exposed to suprainfectious doses of pathogenic Mtb, remain healthy—make antibody responses that are effective in protecting against infection by Mtb. We tested antibodies isolated from 48 healthcare workers and compared these with 12 patients with active TB. We found that antibodies from 7 of 48 healthcare workers but none from active TB patients showed moderate protection against Mtb in an aerosol mouse challenge model. Intriguingly, three of seven healthcare workers who made protective antibody responses had no evidence of prior TB infection by IFN-γ release assay. There was also good correlation between protection observed in vivo and neutralization of Mtb in an in vitro human whole-blood assay. Antibodies mediating protection were directed against the surface of Mtb and depended on both immune complexes and CD4+ T cells for efficacy. Our results indicate that certain individuals make protective antibodies against Mtb and challenge paradigms about the nature of an effective immune response to TB.

Compensatory Mutations of Rifampin Resistance Are Associated with Transmission of Multidrug-Resistant Mycobacterium tuberculosis Beijing Genotype Strains in China

ABSTRACT Mycobacterium tuberculosis can acquire resistance to rifampin (RIF) through mutations in the rpoB gene. This is usually accompanied by a fitness cost, which, however, can be mitigated by secondary mutations in the rpoA or rpoC gene. This study aimed to identify rpoA and rpoC mutations in clinical M. tuberculosis isolates in northern China in order to clarify their role in the transmission of drug-resistant tuberculosis (TB). The study collection included 332 RIF-resistant and 178 RIF-susceptible isolates. The majority of isolates belonged to the Beijing genotype (95.3%, 486/510 isolates), and no mutation was found in rpoA or rpoC of the non-Beijing genotype strains. Among the Beijing genotype strains, 27.8% (89/320) of RIF-resistant isolates harbored nonsynonymous mutations in the rpoA (n = 6) or rpoC (n = 83) gene. The proportion of rpoC mutations was significantly higher in new cases (P = 0.023) and in strains with the rpoB S531L mutation (P < 0.001). In addition, multidrug-resistant (MDR) strains with rpoC mutations were significantly associated with 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat clustering (P = 0.016). In summary, we believe that these findings indirectly suggest an epistatic interaction of particular mutations related to RIF resistance and strain fitness and, consequently, the role of such mutations in the spread of MDR M. tuberculosis strains.

Rifampin Stability in 7H9 Broth and Löwenstein-Jensen Medium

ABSTRACT The objectives of this study were to monitor the stability of rifampin (RIF) in Löwenstein-Jensen medium (L-J medium) and 7H9 broth, which are the media commonly used for drug susceptibility testing (DST) of Mycobacterium tuberculosis. Rifampin degradation in stock solution, 7H9 broth, and L-J medium and during the inspissation process for L-J medium preparation was serially monitored by high-performance liquid chromatography (HPLC). L-J medium-based DST was conducted to examine the effect of L-J medium storage on the DST outcome. The RIF stock solution was stable for at least 3 months when kept at either 4°C or −20°C; RIF in 7H9 broth and L-J medium was almost 50% decayed after 1 week of storage at 37°C, and rifampin could not be detected in 7H9 or L-J medium after 3 weeks or 6 weeks of storage at 37°C. Approximately half of the drug was decomposed after 4 months of storage at 4°C for both media, and after 6 months of storage at 4°C, RIF in L-J medium was undetectable, while 38% of RIF remained in 7H9 medium. Approximately 21, 24, 29, and 35% RIF degradations were detected when the L-J medium was coagulated at 75°C, 80°C, 85°C, and 90°C, respectively. The DST outcomes when using L-J medium stored for different periods of time were consistent with the RIF concentration monitoring data. Rifampin in stock solution is stable for at least 3 months at a reduced storage temperature. Media containing RIF should be prepared strictly according to validated standard operating procedures. RIF degradation is a possible reason for false resistance categorizations of M. tuberculosis isolates in the clinical laboratory.