[Effect of Mycobacterium tuberculosis higBA on Bacterial Stress Response and Intracellular Infection and Immunity].

Objective: To investigate the effect of Mycobacterium tuberculosis ( Mtb) higBA on bacterial stress response and intracellular infection and immunity. Methods: The target gene amplified from Mtb H37Rv genome was cloned to the vector and then transferred to Mycobacterium smegmatis ( Ms) to construct a recombinant strain. Stress response experiment and Raw264.7 mouse macrophage infection was carried out with Ms_higBA, the recombinant strain, and Ms_ vec, the vector strain. Tests were conducted to measure bacterial colony forming unit (CFU) and transcriptional levels of cytokines, including interleukin ( IL)-1ß, IL-6, IL-10, IL-12 p40, interferon ( IFN)- γ, tumor necrosis factor ( TNF)- α, and inducible nitric oxide synthase ( iNOS). Results: The recombinant strain, Ms_higBA, was constructed successfully. According to the findings of the stress response experiment, higBA could indeed enhance bacterial survival under certain conditions of in vitro culture. Intracellular infection experiment demonstrated that higBA enhanced bacterial survival in macrophages and influenced the transcriptional level of cytokines. Conclusion: The higBA genes from Mtb play a role in bacterial stress response and intracellular infection and immunity.

Improvement of the immunogenicity of ESAT-6 via fusion with the dodecameric protein dodecin of Mycobacterium tuberculosis.

Tuberculosis (TB) is a chronic infectious disease that creates a heavy medical burden worldwide. The only approved vaccine, Bacillus Calmette-Guérin (BCG), cannot fully protect adolescents and adults from TB. Therefore, there is an urgent need to develop an effective new vaccine. Previous studies have found that dodecin, a flavin-binding protein of Mycobacterium tuberculosis (Mtb), can form stable dodecamers and has the potential to improve the immunogenicity of Mtb antigens. In this study, we constructed the fusion protein dodecin-ESAT-6 and evaluated the immunogenicity of dodecin, ESAT-6, and dodecin-ESAT-6 separately. Our results showed that dodecin-ESAT-6 is a dodecameric protein that can withstand heat at 95 °C and under SDS-PAGE conditions. Dodecin-ESAT-6 increased the expression of the costimulatory molecules CD80, CD86, and major histocompatibility complex class II (MHC-II) on the surface of RAW264.7 macrophages. Mice immunized with dodecin-ESAT-6 exhibited higher percentages of antigen-specific CD4+ and CD8+ T lymphocytes, higher levels of spleen lymphocyte proliferation and IFN-γ and IL-2 secretion, and a lower level of IL-4 secretion than those immunized with ESAT-6. The IgG, IgG1, and IgG2a titers of the dodecin-ESAT-6 group were significantly higher than those of the ESAT-6 group. Dodecin-ESAT-6 elicited a high IgG2a/IgG1 ratio and tended to produce a predominantly Th1-like response. These results support the conclusion that the dodecin-ESAT-6 dodecameric protein induced strong Th1 immune responses and improved the immunogenicity of ESAT-6, which provides a new strategy for TB vaccine development.

[Preliminary Study on the Secretory Performance of Mycobacterium tuberculosis Hypoxic Response Protein 1 (Rv2626c)].

OBJECTIVE: To verify the secretory ability of the hypoxic response protein 1 (HRP1) encoded by Mycobacterium tuberculosis (Mtb) Rv2626c. METHODS: The target gene attached with His tag was amplified from the genome of Mtb standard virulence strain H37Rv. The recombinant plasmid contained the above amplified product was constructed and electroporated into Mycobacterium smegmatis (Ms) (MC 2155) to construct a recombinant strain. Protein expression was induced under heat condition, and the expression of protein from the culture filtrates and the bacterial lysates was detected afterward. The 10 kDa culture filtrate antigen (CFP-10) (Ms) and CFP-10 (Mtb) were used as positive controls, and the cytoplasmic protein heat shock protein 65 (GroEL2) (Mtb) was used as negative controls. RESULTS: The HRP1, GroEL2 (Mtb), CFP-10 (Mtb) and CFP-10 (Ms) were successfully amplified by PCR from recombinant plasmid, and sequencing results of the recombinant plasmid is right, confirming the successful construction of the recombinant plasmid. The recombinant Ms was successfully constructed and it could express the proteins GroEL2 (Mtb), HRP1, CFP-10 (Mtb) and CFP-10 (Ms). The target protein HRP1 was detected in both of the lysate and the culture filtrate of the recombinant strain by Western blot, which was consistent with the positive control CFP-10. The negative control GroEL2 (Mtb) was only detected in the bacterial lysate, but not detected in the culture filtrate. CONCLUSION: The protein HRP1 encoded by Mtb Rv2626c can be secreted out of Ms by the secretion system of Ms. It may be a secreted protein and play an important role in the pathogenesis of Mtb.

PPE11 of Mycobacterium tuberculosis can alter host inflammatory response and trigger cell death.

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), remains a serious global health problem. The PE/PPE family, featuring unique sequences, structures and expression in Mtb, is reported to interfere with the macrophage response to the pathogen and facilitate its infection. PPE11 (Rv0453) existed in pathogenic mycobacteria and was persistently expressed in the infected guinea pig lungs. However, the role it played in the pathogenesis remains unclear. Here, to investigate the interaction and potential mechanism of PPE11 between pathogens and hosts, we heterologously expressed PPE11 in non-pathogenic, rapidly growing Mycobacterium smegmatis strains. We found that the overexpression of the cell wall-associated protein, PPE11, can improve the viability of bacteria in the presence of lysozyme, hydrogen peroxide and acid stress. Expression of PPE11 enhanced the early survival of M. smegmatis in macrophages and sustained a higher bacterial load in mouse tissues that showed exacerbated organ pathology. Macrophages infected with recombinant M. smegmatis produced significantly greater amounts of interleukin (IL)-1ß, IL-6, tumour necrosis factor (TNF)-α and an early decrease in IL-10 along with higher levels of host cell death. Similar cytokines changes were observed in the sera of infected mice. Accordingly, PPE11 protein causes histopathological changes by disrupting the dynamic balance of the inflammatory factors and promoting host-cell death, indicating a potential role in the virulence of Mtb.

[Optimizing Mycobacterium Recombineering System (pJV53) to Promote the Screening of the Mycobacterium Mutants].

OBJECTIVE: To establish a way for screening Mycobacterium mutants through adding the screening markers into pJV53. METHODS: The sucrose counter selection gene SacB and mutant hygromycin-resistant gene hygS were inserted into pJV53; The recovery of the hygromycin-resistance indicated the successful homologous recombination in Mycobacterium smegmatis (Ms), which could serve as mutant screening marker; The sucrose counter selection could be used to screen the plasmid-free mutants. RESULTS: The recombinant plasmid pJV53-SacB-hygS were successfully constructed. The rifampin-resistant rpoB D516Y and rpoB H526Q mutants and MSMEG_4487 G188A mutant were efficiently screened out. All mutants had shed the plasmid successfully. CONCLUSION: pJV53-SacB-hygS can efficiently contribute to construct and screen the mutants and to get the mutants shedding the plasmid self, which has high value of extensive application; the D516Y and H526Q mutations in gene rpoB of Mycobacterium tuberculosis contribute to its rifampin-resistance.

[Mycobacterium Tuberculosis Rv3084 Encodes Functional Esterase and Suppresses the Pro-inflammatory Cytokines in vivo].

OBJECTIVE: To explore the biological characteristics of the esterase LipR encoded by Mycobacterium tuberculosis (MTB) Rv3084 and its immunomodulatory function in vivo. METHODS: The LipR gene was amplified from MTB H37Rv strain to construct recombinant expression plasmid. After sequencing, the recombinant plasmid was transformed into E. coli for expression and purification of LipR protein. The expressed protein was confirmed with Western blot assay. The hydrolyzing activity of LipR was detected and the factors affecting LipR enzyme activity were analyzed. Mice were intramuscularly injected with 0.1 mL (containing plasmid DNA 100 µg) recombinant eukaryotic plasmid three times (day 1, 8, and 15); seven days after the last injection, the mice were executed, and the lung and spleen were taken for cytokine detection. RESULTS: The recombinant expression plasmid was successfully constructed and it was found that LipR protein was mainly expressed in the form of inclusion bodies in E. coli with the relative molecular mass of about 33×10 3. LipR was demonstrated as an alkaline eurythermic esterase, due to the preference of hydrolyzing short carbon chain esters with optimal hydrolyzing activity on pNP-acetate (pNPA, C2) and the capability in tolerance of high pH and temperature; in the presence of different detergents or metal ions, the activity of LipR hydrolyzing pNP-butyrate (pNPB, C4) was inhibited to some extent. In the mouse model, it was found that LipR could inhibit the secretion of interferon-γ (IFN- γ) and interleukin-2 (IL-2), but to stimulate the secretion of IL-10. CONCLUSION: The esterase LipR may be one of the esterases help M. tuberculosis withstand harsh environment inside the host in collaboration, and simultaneously act as an immune modulator to inhibit the secretion of pro-inflammatory cytokines and consequently impact the killing effect of host immune system against M. tuberculosis.

[Effect of the Expression of iNOS Induced by Mycobacterium tuberculosis CRISPR-associated Csm4 (Rv2820c) on Intracellular Viability of Mycobacterium smegmatis].

OBJECTIVE: To determine how Csm4 protein expression affects intracellular survival of Mycobacterium smegmatis(MS). METHODS: Csm 4 gene was amplified by PCR to construct pMV261-Csm4 shuttle expression plasmid. The Csm4 protein expression in MS_Csm4 was detected by Western blot after electroporation of the recombinant plasmid into MS. The growth kinetics of MS_Csm4 in vitro and the influence of reactive N,O species on the growth of MS_Csm4were observed. The intracellular survival of MS_Csm4 and expressions of inducible nitric oxide synthase gene (iNOS) and nitric oxide production (NO) were detected after infection with THP-1 macrophages. RESULTS: Csm4 protein was successfully expressed in MS_Csm4,which did not affect the growth of the recombinant MS. Reactive N,O species decreased MS_Csm4 colony forming unit (CFU) in vitro. THP-1 increased the expression of iNOS and NO production and decreased intracellular survival of MS_Csm4. CONCLUSION: Recombinant MS_Csm4 is susceptible to reactive N,O species in vitro. THP-1 promotes NO release and thus discourages intracellular survival of MS.

Double mutation in DNA gyrase confers moxifloxacin resistance and decreased fitness of Mycobacterium smegmatis.

Objectives: Ofloxacin and moxifloxacin are the most commonly used fluoroquinolones (FQs) for the treatment of tuberculosis. As a new generation FQ, moxifloxacin has been recommended for the treatment of ofloxacin-resistant TB. However, the mechanism by which ofloxacin-resistant Mycobacterium tuberculosis further gains resistance to moxifloxacin remains unclear. Methods: We used Mycobacterium smegmatis as a model for studying FQ resistance in M. tuberculosis . Moxifloxacin-resistant M. smegmatis was selected in vitro based on strains with primary ofloxacin resistance. The gyrA and gyrB genes of the resistant strains were sequenced to identify resistance-associated mutations. An in vitro competition assay was applied to explore the influence of gyrA / gyrB mutations on bacterial fitness. Finally, we evaluated the clinical relevance of our findings by analysing the WGS data of 1984 globally collected M. tuberculosis strains. Results: A total of 57 moxifloxacin-resistant M. smegmatis strains based on five ofloxacin-resistant strains were obtained. Sequencing results revealed that all moxifloxacin-resistant strains harboured second-step mutations in gyrA or gyrB . The relative fitnesses of the double-mutation strains varied from 0.65 to 0.93 and were mostly lower than those of their mono-mutation parents. From the genomic data, we identified 37 clinical M. tuberculosis strains harbouring double mutations in gyrA and/or gyrB and 36 of them carried at least one low-level FQ-resistance mutation. Conclusions: Double mutation in DNA gyrase leads to moxifloxacin resistance and decreased fitness in M. smegmatis . Under current dosing of moxifloxacin, double mutations mainly happened in M. tuberculosis strains with primary low-level resistance mutations.

Mycobacterium tuberculosis PE25/PPE41 protein complex induces activation and maturation of dendritic cells and drives Th2-biased immune responses.

Mycobacterium tuberculosis evades innate host immune responses by parasitizing macrophages and causes significant morbidity and mortality around the world. A mycobacterial antigen that can activate dendritic cells (DCs) and elicit effective host innate immune responses will be vital to the development of an effective TB vaccine. The M. tuberculosis genes PE25/PPE41 encode proteins which have been associated with evasion of the host immune response. We constructed a PE25/PPE41 complex gene via splicing by overlapping extension and expressed it successfully in E. coli. We investigated whether this protein complex could interact with DCs to induce effective host immune responses. The PE25/PPE41 protein complex induced maturation of isolated mouse DCs in vitro, increasing expression of cell surface markers (CD80, CD86 and MHC-II), thereby promoting Th2 polarization via secretion of pro-inflammatory cytokines IL-4 and IL-10. In addition, PE25/PPE41 protein complex-activated DCs induced proliferation of mouse CD4(+) and CD8(+) T cells, and a strong humoral response in immunized mice. The sera of five TB patients were also highly reactive to this antigen. These findings suggest that interaction of the PE25/PPE41 protein complex with DCs may be of great immunological significance.

The Mycobacterium bovis BCG prime-Rv0577 DNA boost vaccination induces a durable Th1 immune response in mice.

Tuberculosis remains a major global health problem and effective vaccines are urgently needed. In this study, we used the combined DNA- and protein-based vaccines of immunodominant antigen Rv0577 to boost BCG and evaluated their immunogenicity in BALB/c mice. Our data suggest that the booster vaccine may substantially enhance the immunogenicity of BCG and strengthen both CD4+ T cell-mediated Th1 and CD8+ T cell-mediated cytolytic responses. Compared with the protein-based vaccine, the DNA-based vaccine can induce more durable Th1 immune response, characterized by high levels of antibody response, proliferation response, percentages of CD4+/CD8+ and cytokine secretion in antigen-stimulated splenocyte cultures. In conclusion, we for the first time, developed a protein- and plasmid DNA-based booster vaccine based on Rv0577. Our findings suggest that antigen Rv0577-based DNA vaccine is immunogenic and can efficiently boost BCG, which could be helpful in the design of an efficient vaccination strategy against TB.

[Research on the relevance between the virulent genes differential expression and pathogenecity of Leptospira with microarray].

OBJECTIVE: To find the change of virulent gene expression and to analyze the relevance between the virulent change and the gene expression. METHODS: Grouped guinea pigs were inoculated with 1 mL Leptospira cultured in vivo, Leptospira cultured in vitro and the Leptospira culture medium through abdominal subcutaneous respectively. The survival rate, body mass and temperature change of guinea pigs in different groups were measured within 15 d after the inoculation, then the survived guinea pigs were scarified, and the organ coefficient was also measured to know the virulence of Leptospira cultured in different environment. The amplified gene segments from Leptospira were used as probes and wrote the microarray. The total RNA was extracted from Leptospira standard strain cultured in culture medium and guinea pigs. After reverse transcription to cDNA, they were labeled with Cy3 and Cy5 respectively. Labeled cDNA was mixed and hybridized with the microarray. The hybridized mircroarray was scanned and analysed. RESULTS: The survival rate of inoculated guinea pig was different from group to group (in vivo group: 0%; in vitro group: 88.9%; culture medium group: 100%). The guinea pigs in vivo group had a higher temperature (P<0.05), lighter body mass (P<0.05), larger organ coefficient (P<0.05) and a more serious hemorrhage in lung. The genes from Leptospira: LA1027, LA1029, LA4004, LA3050, LA3540, LA0327, LA0378, LA1650, LA3937, LA2089, LA2144, LA3576, LA0011 and gene of Loa22 were up regulation after continuously cultured in guinea pigs. CONCLUSION: The pathogenic ability of Leptospira cultured in different environment is different and the gene expression of Leptospira is different between in vivo and in vitro as well. The understanding of the meaning of this change might help to know the pathogenecity of Leptospira.

[Prokaryotic Expression of Rv0757 Gene in Mycobacterium tuberculosis and the Effect of Its Coding Protein on the Function of Macrophage].

OBJECTIVE: To construct recombinant plasmid pET28a-Rv0757 with Mycobacterium tuberculosis (Mtb) Rv0757 gene, and to determine the effect of the protein of Rv0757 gene on ANA-1 cells. METHODS: We recombined the amplified Rv0757 gene into theprokaryotic plasmid pET28a (+). The expressed product was identified by SDS-PAGE and Western blot. Murine macrophages were treated with purified protein PhoP. Survived cells, lactic dehydrogenase (LDH), nitric oxide (NO), and cell apoptosis were detected after the treatment. RESULTS: We successfully constructed the recombinant plasmid pET28a-Rv0757. The target protein was confirmed by SDS-PAGE and Western blot. The protein had no significant effect on cell numbers and LDH activities in the culture supernatant, but it inhibited the release of NO and apoptosis of ANA-1 cells. CONCLUSION: pET28a-Rv0757 plasmid with prokaryotic expression was successfully constructed. The targetprotein has no toxicity on macrophages, but it can inhibit NO release and cell apoptosis of ANA-1.

[The cytotoxic effect and the effect of permeability with OmpA like protein Loa22 from Leptospira interrogans serovar].

OBJECTIVE: To study the toxic effect and the change of permeability on human umbilical vein endothelia (HUVE) of the Loa22 protein from virulent serovar Lai. Leptaspira interrogans by expressing its protein. METHODS: In this study, the pGEX-Loa22 peptide prokaryotic recombinant plasmid of Leptospira interrogans serovar Lai preserved in our laboratory was used to express Loa22 fusion protein with GST lable. Then the target fusion protein was obtained by using affinity chromatography with the GST-Trap FF Column. The purified Loa22 fusion protein was detected by SDS-PAGE and confirmed by Western blot assay using the mouse anti-GST tag monoclonal anti-body. pGEX-Loa22 protein was administered to culture with human umbilical vein endothelial cells (HUVEC) to elucidate the cytotoxic role and the change of permeability of leptospiral outer membrane proteins. RESULTS: The recombiant plasmid with Loa22 mature peptide was expressed successfully and the protein was purfied. Significant higher level of apoptosis ratio, lower CCK-8 aborntion, and increasing permeability on HUVEC were observed after treated the HUVEC with the expressed fusion protein. CONCLUSION: The purified Loa22 fusion protein have obvious toxic effects on vascular endothelial cells, and also it can increase permeability of HUVEC.

Enhancement of antimycobacterial Th1-cell responses by a Mycobacterium bovis BCG prime-protein boost vaccination strategy.

Tuberculosis is a major global health problem, and the only available vaccine Bacille Calmette-Guérin (BCG) is not sufficiently effective against the disease. It is extremely urgent to develop novel vaccine approaches. Previous research demonstrated that there were several Regions of Difference (RD1-16) between the substrains of BCG and Mycobacterium tuberculosis or Mycobacterium bovis. The ORFs Rv1769 and Rv1772 are located in the RD14 deletions and have not been major targets of study. However, some studies have demonstrated that the two genes (Rv1769 and Rv1772) are excellent T cell antigens, which might induce an immune response. What kind of role these ORFs might play in anti-mycobacterial immunity, however, is still unknown. In our research we used the BCG prime-protein boost strategy to immunize BALB/c mice and evaluated its immunogenicity. Our data suggest that our novel BCG-P+PRO69 vaccine could elicit the most long-lasting and strongest Th1 type cellular immune responses. This response is characterized by a strong antibody response, the proliferation rate of splenocytes, a high percentage of CD4+ and CD8+ T cells and high levels of IFN-γ in antigen-stimulated splenocyte cultures. These results indicate that prime-boost is a potent strategy and the protein of gene Rv1769 is a potential antigen or subunit vaccine to TB for further study.

[Research on the expression of hemolysin genes of Leptospira in vivo by genechip].

OBJECTIVE: To explore the expression of hemolysin genes of Leptospira in infected host. METHODS: Amplified the gene segment of hemolysin genes from the genome of Leptospira by PCR for gene probe. Manufacture genechip by the VersArray Chipwriter systerm. The total RNAs of Leptospira before and after infection host were extracted, reversely transcribed to cDNA, after the random PCR, the products were marked with HEX and CY5 respectively, and hybridized to genechip to demonstrate the expression of hemolysin genes of Leptospira. RESULTS: The hemolysin genes LA1029 (Ratio = 0.65), LA1027 (Ratio = 0.53) were up-regulated after infection of host; LA3540 (Ratio = 1.88), LA3937 (Ratio = 5.58), LA1029 (Ratio = 3.00) were up-regulated and LA4004 (Ratio = 0.67) was down-regulated in live than in blood; LA3937 (Ratio = 2.28), LA1029 (Ratio = 2.20) were up-regulated in kidney than in blood. CONCLUSION: The expression level of hemolysin genes exist observable differences with inducement in vivo and in different organs. These suggested that these genes are probably involved in the pathogenesis and and disease progression.

[Progress in research on molecular biology and application in dominant antigens ESAT6 and CFP10 of TB vaccine].

As the dominant antigens, early secreted antigenic target 6 (ESAT6, E6) and culture filtrate protein 10 (CFP10, C10) had once been the focus of tuberculosis (TB) vaccine due to their capability of inducing strong cell immune response in the host. They are also endowed with promising future of prevention against and diagnosis of TB. In this review, we systematically introduce recent research progress of E6 and C10, especially in structure-function, biological characteristics, protein expression and secretion, host immunity and vaccine development, and the prospects of their application are also discussed.

Evaluation of immunogenicity and protective efficacy against Mycobacterium tuberculosis infection elicited by recombinant Mycobacterium bovis BCG expressing human Interleukin-12p70 and Early Secretory Antigen Target-6 fusion protein.

ESAT-6 protein of Mycobacterium tuberculosis is absent in Mycobacterium bovis BCG and Mycobacterium microti and has been demonstrated to stimulate strong cell-mediated immunity. IL-12 can play crucial roles in regulating IFN-γ production and Th1 effectors production. In this study, we constructed three rBCG vaccines that could express proteins of human IL-12p70 and/or ESAT-6 and evaluated their immunogenicity and protective efficacy in mice. Our experiments illustrated that the rBCG-IE (expressing a fusion protein of human IL-12p70 and ESAT-6) was capable of inducing stronger Th1 type cell-mediated immune responses than conventional BCG, or rBCG-I (expressing human IL-12p70), or rBCG-E (expressing ESAT-6). However, the results of protective experiments showed that rBCG-IE could only confer similar and even lower protective efficacy against M. tuberculosis H37Rv infection compared with BCG vaccine.

A novel recombinant Mycobacterium bovis bacillus Calmette-Guerin strain expressing human granulocyte macrophage colony-stimulating factor and Mycobacterium tuberculosis early secretory antigenic target 6 complex augments Th1 immunity.

Since Mycobacterium bovis bacillus Calmette-Guerin strain (BCG) fails to protect adults from pulmonary tuberculosis (TB), there is an urgent need for developing a new vaccine. In this study, we constructed a novel recombinant BCG strain (rBCG) expressing human granulocyte macrophage colony-stimulating factor (GM-CSF) and the 6 kDa early secretory antigenic target (ESAT6) of Mycobacterium tuberculosis, named rBCG:GE (expressing GMCSF-ESAT6 complex), and evaluated the immunogenicity of the construct in BALB/c mice. Our results indicated that the rBCG:GE was able to induce higher titer of antibody than the conventional BCG, the rBCG:G (expressing GM-CSF) and the rBCG:E (expressing ESAT6). Moreover, the rBCG:GE also elicited a longer-lasting and stronger Th1 cellular immune responses than the other groups, which was confirmed by the incremental proliferation of splenocytes, the increased percentages of CD4(+) and CD8(+) T cells of spleen, the elevated level of interferon-γ in splenocyte culture after tuberculin-purified protein derivative stimulation, and the increased concentration of GM-CSF in serum. The data presented here suggested the possibility that the recombinant BCG:GE might be a good vaccine candidate to TB.

[The OmpA-like protein Loa22 from Leptospira interrogans serovar lai induces apoptosis in A549 via Ca2+ signal pathway].

OBJECTIVE: To study the effect of Loa22 mature peptide on the apoptosis of A549 to explore the mechanism of pulmonary impairment in severe forms of leptospirosis. METHODS: Loa22 mature peptide (100 microg/mL) was administered to culture with human lung adenocarcinoma cell line (A549). After 24 hours, the apoptosis, the concentration of calcium of the cells were evaluated. The F-actin cytoskeleton structure was observed and calmdulin (CaM) mRNA expression was also detected. At the same time, after the pretreatment of A549 with PLC specific inhibitor U73122, adding an appropriate amount of mature peptide of Loa22 to act on the cells for a period of time, then detection same index. RESULTS: Loa22 mature peptide could induce the increase of intracellular Ca2+ concentration ([Ca2+]i), CaM expression in mRNA level, the activity of LDH, and cytoskeleton rearrangement of F-actin. But after blocking the signal pathway of PLC, Loa22 mature peptide reduced the increase degree of [Ca2+]i, apoptosis rate, the expression of CaM mRNA, the activity of LDH compared with the unblocked group. CONCLUSION: These data suggest that Loa22 mature peptide involves in the pathological processes of L. interrogans invasion and increase apoptosis in A549 by increase of [Ca2+]i through signaling pathway of PLC.

Compensatory effects of M. tuberculosis rpoB mutations outside the rifampicin resistance-determining region.

Mycobacterium tuberculosis has been observed to develop resistance to the frontline anti-tuberculosis drug rifampicin, primarily through mutations in the rifampicin resistance-determining region (RRDR) of rpoB. While these mutations have been determined to confer a fitness cost, compensatory mutations in rpoA and rpoC that may enhance the fitness of resistant strains have been demonstrated. Recent genomic studies identified several rpoB non-RRDR mutations that co-occurred with RRDR mutations in clinical isolates without rpoA/rpoC mutations and may confer fitness compensation. In this study, we identified 33 evolutionarily convergent rpoB non-RRDR mutations through phylogenomic analysis of public genomic data for clinical M. tuberculosis isolates. We found that none of these mutations, except V170F and I491F, can cause rifampin resistance in Mycolicibacterium smegmatis. The compensatory effects of five representative mutations across rpoB were evaluated by an in vitro competition assay, through which we observed that each of these mutations can significantly improve the relative fitness of the initial S450L mutant (0.97-1.08 vs 0.87). Furthermore, we observed that the decreased RNAP transcription efficiency introduced by S450L was significantly alleviated by each of the five mutations. Structural analysis indicated that the fitness compensation observed for the non-RRDR mutations might be achieved by modification of the RpoB active centre or by changes in interactions between RNAP subunits. Our results provide experimental evidence supporting that compensatory effects are exerted by several rpoB non-RRDR mutations, which could be utilized as additional molecular markers for predicting the fitness of clinical rifampin-resistant M. tuberculosis strains.