利用无传染性耻垢分枝杆菌建立小鼠结核病模型的探索

目的:利用耻垢分枝杆菌(M.smegmatismc2155)建立C57BL/6小鼠结核病模型。方法:每天以高剂量(5×107CFU)耻垢分枝杆菌给C57BL/6小鼠腹腔注射,连续感染4周,检测耻垢分枝杆菌对小鼠的致病性。分别于2周和4周处死小鼠,无菌条件下解剖小鼠取肺、脾脏组织匀浆,进行组织内细菌活力检测;通过嗜酸性染色进行分枝杆菌的鉴定;同时进行病理切片的制备,观察肺和脾脏组织的病理变化;最后进行菌体DNA的提取和基因检测,根据上述指标确定小鼠结核病模型的建立是否成功。结果:腹腔感染小鼠2周后,模型组小鼠只有脾脏组织匀浆液出现抗酸染色阳性菌落,肺部组织未见阳性菌落。腹腔感染小鼠4周后,模型组小鼠肺、脾脏组织匀浆液中均可见大量抗酸染色阳性的菌落;组织病理学观察结果显示:小鼠肺组织主要表现为以中性粒细胞为主的炎性病变;基因检测结果表明:模型组小鼠肺组织匀浆液中可检测到耻垢分枝杆菌特异性3-磷酸甘油醛脱氢酶(gap)基因,而脾脏组织未扩增出耻垢分枝杆菌特异性基因。结论:通过腹腔注射无致病性耻垢分枝杆菌方法,成功建立C57BL/6小鼠结核病发生模型,为结核分枝杆菌与宿主相互作用研究提供安全的疾病模型。     

Ⅱ型糖尿病并发结核病小鼠模型的建立及评价

目的建立Ⅱ型糖尿病并发结核病小鼠模型,对其肺组织病变过程进行评价。方法高脂高糖饲料喂养C57BL/6J小鼠,腹腔注射STZ,建立Ⅱ型糖尿病小鼠模型。采用滴鼻的方式将结核分枝杆菌H37Rv接种至糖尿病小鼠体内,建立糖尿病并发结核病小鼠模型。分别于感染后的1~8周、10周及12周解剖小鼠,肉眼观察小鼠肺脏病变情况,活菌菌落计数,肺组织行HE及抗酸染色,镜下观察病理学改变。结果肉眼观察发现,随着感染时间的增长肺部感染情况逐渐加重,至第8周时病灶达到全肺的80%,第10周时病变范围开始缩小;感染8周后HE染色肺组织出现典型的肉芽肿结构,至第10周部分病变肺组织出现修复现象;抗酸染色可见结核分枝杆菌。结论从大体病变、菌落计数及病理改变等方面对小鼠模型进行评价,发现糖尿病并发结核病小鼠模型较单一的结核病小鼠模型具有病变出现早,病程进展快等特点,与临床患者的某些阶段表现类似,因此实验构建的糖尿病并发结核病小鼠模型基本成功。     

小鼠结核分枝杆菌耐药模型的建立与评价

目的 建立小鼠结核分枝杆菌耐利福平株静脉感染小鼠模型.方法 30只BALB/c雌性小鼠经尾静脉感染结核分枝杆菌耐利福平株每只106 CFU,观察小鼠一般状况,并分别在感染后6周、10周、14周处死小鼠,进行脾、肺组织病理切片、抗酸染色、计脏器荷菌数.结果 感染后小鼠体重呈逐渐上升趋势,脏器病理变化随时间推移由急性炎症逐...     

人白细胞介素12表达质粒与Mtb8.4基因疫苗 联合免疫增强对小鼠结核杆菌感染的免疫保护效果

本课题旨在研究结核分枝杆菌Mtb8.4基因疫苗与人白细胞介素12（hIL-12）联合免疫小鼠所诱导的细胞免疫应答及对小鼠结核杆菌感染的免疫保护效果。40只C57BL/6N小鼠随机分为Mtb8.4基因疫苗＋hIL-12质粒组（联合免疫组）、Mtb8.4基因疫苗组、卡介苗（BCG）组、空载体组和PBS组,基因疫苗、空载体和PBS,经肌内注射法免疫各组小鼠,每隔3周免疫1次,共免疫3次,BCG组经尾部皮下注射1×106 CFU BCG免疫1次。免疫4周后,每组处死3只小鼠,采用酶联免疫吸附法（ELISA）检测脾细胞培养上清中细胞因子水平;乳酸脱氢酶（LDH）释放法检测细胞毒性T细胞（CTL）杀伤活性。每组其余5只小鼠用结核杆菌H37Rv强毒株经尾静脉攻击,4周后,计数肺和脾组织中的结核杆菌菌落数,对小鼠部分肺和脾组织作病理切片,HE染色观察组织病变程度,Z-N染色查抗酸杆菌,观察该疫苗对小鼠结核杆菌感染的免疫保护效果。结果显示,联合免疫组能诱导较强的抗原特异性Th1型细胞免疫应答,免疫小鼠脾细胞培养上清液IFN-γ和IL-2水平（分别为1493.34±8.128pg/mL、747.489±48.676pg/mL）,显著高于Mtb8.4基因疫苗组,与BCG组相当,IL-4分泌减少,特异性CTL杀伤活性增强,对小鼠结核杆菌感染有较好的免疫保护效果,使小鼠肺和脾组织中的结核杆菌菌落数显著减少,组织病变明显减轻,其效果与卡介苗（BCG）组相当,优于Mtb8.4基因疫苗组。表明hIL-12表达质粒与Mtb8.4基因疫苗联合免疫后,能够增强Mtb8.4基因疫苗所诱导的细胞免疫应答,使Mtb8.4基因疫苗的免疫效力得到很大提高。     

结核分枝杆菌持续感染小鼠模型的建立及其免疫特征

为建立小鼠结核分枝杆菌持续感染模型并研究其免疫应答特征,选取雌性C57BL/6小鼠,经尾静脉感染结核分枝杆菌H37Rv株,并以异烟肼和利福平联合治疗。分别于感染后4、8、12周处死小鼠,用平板法计数肺和脾荷菌数,酶联免疫吸附试验(ELISA)检测血清中特异性抗体水平及亚类,流式细胞术检测CD4+脾淋巴细胞经结核分枝杆菌抗原——纯化蛋白衍生物(PPD)刺激后分泌细胞因子的细胞比例。结果显示,感染4周后小鼠肺和脾荷菌数lg CFU分别达3.67±0.25和3.54±0.24,至少持续8周;药物治疗可有效降低脏器荷菌数。感染12周后,感染组血清中抗结核分枝杆菌特异性抗体水平显著升高(P0.01),且以IgG1为主;治疗组总IgG抗体水平显著低于感染组(P0.01),且IgG2a相对较高(P0.05)。感染组CD4+脾淋巴细胞中γ干扰素(IFN-γ)和白细胞介素2(IL-2)分泌细胞比例显著增加(P0.01和P0.001),而IL-4分泌细胞比例显著降低(P0.01);治疗组IL-2和IL-4分泌细胞比例显著低于正常组(P0.05和P0.01)。本研究建立的小鼠结核分枝杆菌持续感染模型有望用于结核病治疗性疫苗和药物的研发及筛选。     

铜绿假单胞菌重组Bb-OprI疫苗免疫及PA01株感染后小鼠脾细胞因子IL-17和Treg的变化

目的:研究铜绿假单胞菌重组Bb-OprI疫苗免疫及PA01株攻击后小鼠产生的保护力,及脾细胞因子IL-17和Treg的变化。方法:将5×10~9CFU的Bb-Opr I疫苗灌胃接种BALB/c鼠,每周3次,连续3周;于初次接种后4周,将5×10~6CFU的PA01株滴鼻攻击小鼠,攻击后2周处死小鼠,分离肺组织和脾脏,培养肺组织的细菌并进行菌落计数,以铜绿假单胞菌抗原(PaAg)诱导培养脾细胞,PCR扩增IL-17和Foxp3基因。结果:Bb-Opr I疫苗组小鼠的肺组织细菌计数(0.46±0.09)×10~8CFU明显低于空载体对照组(5.42±0.79)×10~8CFU和Bb对照组(6.20±0.95)×10~8CFU(P0.01),从疫苗组小鼠的脾细胞DNA可扩增出380 bp的IL-17和250 bp的Foxp3基因。结论:重组Bb-OprI疫苗可诱导小鼠产生Th17和Treg免疫应答,从而抵抗PA01株感染并维持免疫稳态。     

结核感染小鼠肺部病理变化与T细胞免疫关系的实验研究

目的探讨结核病小鼠肺部病理变化与T细胞亚群的关系。方法将60只C57BL小鼠随机分为3组,每组20只。结核病模型组小鼠经球后静脉丛注射人型结核分枝杆菌标准毒力珠（H37RV）;免疫调节剂组结核病模型小鼠于H37RV感染后第3、10、17天分别肌注22.5μg母牛分枝杆菌菌苗（微卡菌苗）;正常对照组小鼠未做处理。感染4周后,留取外周血用流式细胞仪测定T细胞亚群;取肺组织,采用常规苏木精-伊红染色和抗酸染色后病理切片观察病理变化。结果结核病模型组小鼠大部分肺组织实变,多数肺泡腔消失,肺泡隔重度增宽,肺间质内可见大量炎性细胞浸润,浸润细胞以中性粒细胞为主,淋巴细胞较少。残存的肺泡腔中有红细胞、纤维素渗出。肺实变区内分布大量的紫红色结核分枝杆菌,主要存在于单核-巨噬细胞胞质内。外周血T细胞亚群的相对比率明显减少。免疫调节剂组肺组织病变和结核病模型组大致相似,但实变病灶较少,多数肺泡腔存在,肺泡隔内浸润淋巴细胞较多,肺泡腔内有渗出单核-巨噬细胞。肺实变区内结核分枝杆菌明显少于结核病模型组,CD3^＋与CD4^＋T细胞比例增加,能显著增加γδT细胞比例。结论微卡菌苗可增强结核病小鼠的免疫功能,使肺实变病灶较少和病灶内结核分枝杆菌明显减少,肺泡腔内单核-巨噬细胞增多,为免疫调节剂辅助治疗结核病提供理论依据。     

结核分枝杆菌感染对NLRP3炎性小体活化的调控作用研究进展

结核病(tuberculosis, TB)是一种主要由结核分枝杆菌(Mycobacterium tuberculosis,Mtb)感染引起的世界范围内影响广泛的细菌性疾病。巨噬细胞通过细胞表面表达的多种受体感知进而吞噬Mtb。Mtb侵染巨噬细胞时,ESX-1等分泌系统会激活以NLRP3为代表的炎性小体,并促进下游IL-18和IL-1β的活化和分泌。同时Mtb也可以通过Zmp1、Ptp A等方式抑制NLRP3炎性小体活化,使Mtb得以在宿主细胞内存活和繁殖。揭示NLRP3炎性小体在Mtb感染中的作用及活化机制,可为结核病的预防和治疗提供参考。     

结核病防治基础研究进展

结核病(tuberculosis)是由结核分枝杆菌(Mycobacterium tuberculosis,Mtb)引起的一种慢性传染病.结核病经呼吸道传播,肺部是Mtb感染的主要和起始器官.因此,肺结核是最主要的结核病.除肺部外,其他系统器官如骨关节、淋巴结、泌尿生殖系统、肠道、肝脏等均可受累而发生结核病.     

结核分枝杆菌感染小鼠的脾脏和肺脏组织荷菌量与病理变化

目的分析结核急性感染小鼠脾脏和肺脏的组织荷菌量以及病理的动态变化。方法以3×105CFU结核分枝杆菌标准株H37Rv尾静脉途径感染雌性C57BL/6J小鼠,测定感染后1 d、1周、2周、3周、4周、6周和8周肺脏及脾脏组织的荷菌量,脾脏和肺脏组织经HE染色分析病理变化。结果感染动物的脾脏荷菌量在前3周逐步提高,在4～8周脾脏荷菌量维持稳定,肺脏组织在2～8周组织荷菌量逐步升高。病理分析显示动物感染3周后肺脏和脾脏出现肉芽肿,在6～8周病理损伤加重。结论小鼠经尾静脉感染高剂量结核分枝杆菌在8周前表现为急性感染状态,适用于抗结核药物的体内药效学评价。     

Membrane TNF confers protection to acute mycobacterial infection

Background

Tumour necrosis factor (TNF) is crucial for the control of mycobacterial infection as TNF deficient (KO) die rapidly of uncontrolled infection with necrotic pneumonia. Here we investigated the role of membrane TNF for host resistance in knock-in mice with a non-cleavable and regulated allele (mem-TNF).

Methods

C57BL/6, TNF KO and mem-TNF mice were infected with M. tuberculosis H37Rv (Mtb at 100 CFU by intranasal administration) and the survival, bacterial load, lung pathology and immunological parameters were investigated. Bone marrow and lymphocytes transfers were used to test the role of membrane TNF to confer resistance to TNF KO mice.

Results

While TNF-KO mice succumbed to infection within 4–5 weeks, mem-TNF mice recruited normally T cells and macrophages, developed mature granuloma in the lung and controlled acute Mtb infection. However, during the chronic phase of infection mem-TNF mice succumbed to disseminated infection with necrotic pneumonia at about 150 days. Reconstitution of irradiated TNF-KO mice with mem-TNF derived bone marrow cells, but not with lymphocytes, conferred host resistance to Mtb infection in TNF-KO mice.

Conclusion

Membrane expressed TNF is sufficient to allow cell-cell signalling and control of acute Mtb infection. Bone marrow cells, but not lymphocytes from mem-TNF mice confer resistance to infection in TNF-KO mice. Long-term infection control with chronic inflammation likely disrupting TNF mediated cell-cell signalling, additionally requires soluble TNF.     

Mycobacterium tuberculosis infects dendritic cells with high frequency and impairs their function in vivo

Mycobacterium tuberculosis (Mtb) is thought to reside in macrophages, although infected dendritic cells (DCs) have been observed. Thus, although cellular associations have been made, global characterization of the cells harboring Mtb is lacking. We have performed temporal and quantitative characterization of the cells harboring Mtb following aerosol infection of mice by using GFP-expressing bacteria and flow cytometry. We discovered that Mtb infects phagocytic cells of diverse phenotypes, that the predominant infected cell populations change with time, and that myeloid DCs are the major cell population infected with Mtb in the lungs and lymph nodes. We also found that the bacteria in the lung-draining lymph node are transported there from the lungs by a CCL19/21-dependent mechanism and that the transport of bacteria to the lymph node is a transient phenomenon despite chronic infection. In addition, we found that the lymph node cell subsets that are most efficacious in stimulating Mtb-specific, TCR-transgenic CD4(+) T lymphocytes are not infected with the bacteria and are scarce or absent from the lungs of infected mice. Finally, we found that the lung cell populations that are infected with Mtb at high frequency are relatively ineffective at stimulating Ag-specific CD4(+) T lymphocytes, and we have obtained evidence that live Mtb can inhibit MHC class II Ag presentation without a decrease in the surface expression of MHC class II. These results indicate that Mtb targets DC migration and Ag presentation in vivo to promote persistent infection.     

气雾攻击法感染结核病小鼠模型的建立及其综合评价

目的 模拟自然感染方式建立结核病小鼠模型,并对其病理变化进行综合评价.方法 通过气雾攻击方式将结核分枝杆菌H37Rv接种至C57BL/6J小鼠体内.在感染后的4周、6周、8周对小鼠进行micro-CT活体动态扫描,无菌分离肺脏和脾脏,肉眼观察病变情况,活菌菌落计数,组织病理检测(HE和抗酸染色).结果 肉眼观察和micro-CT扫描发现,不同时间小鼠肺部感染情况逐渐加重,至感染后第8周时病变弥漫至整个肺部;HE染色肺组织出现弥漫性肉芽肿样实变;抗酸染色可见结核分枝杆菌.结论 通过大体病变、病理、影像、菌落计数几个方面对建立的小鼠模型进行综合分析,证明利用气雾攻击法感染的结核病小鼠模型建立成功;该模型在形成病变时与结核患者的情况存在一定差异,对其完善的综合评价有助于在相关研究中对该小鼠模型的合理应用.     

Protective and survival efficacies of Rv0160c protein in murine model of Mycobacterium tuberculosis

The proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) multi-gene families code for approximately 10 % of the Mycobacterium tuberculosis (Mtb) genome. These proteins are thought to be virulence factors that participate in impounding the host immune responses. While some members have been studied, the functions of most PE/PPE proteins are yet to be explored. The studies presented here have specifically characterized the roles of one of the PE proteins of Mtb, Rv0160c (PE4), in mycobacterial persistence and in prophylactic efficacy. We have expressed Rv0160c in a non-pathogenic fast-growing Mycobacterium smegmatis strain and demonstrated that the protein improves the survival of mycobacteria in macrophages and in mice. The protein has also shown its effect under physiological stress of bacteria, as evidenced by elevated expression in acidic and in hypoxic conditions. In mice, the level of Rv0160c was noticeably high during the chronic stage of tuberculosis. The seroreactivity of the protein against different categories of tuberculosis patients revealed a strong B-cell humoral response in freshly infected pulmonary tuberculosis patients. In mice, it exhibited increased IL-2, TNF, and IL-6 production. The antigenic properties of the protein directed towards the protective efficacy against the Mtb challenge. All together, our findings have identified Rv0160c as an in vivo expressed immunodominant antigen which plays a crucial role in the pathogenesis of mycobacterial disease and could prove to be a good preventive antigen for tuberculosis.     

Discovery of novel 5-(ethyl or hydroxymethyl) analogs of 2'-'up' fluoro (or hydroxyl) pyrimidine nucleosides as a new class of Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium inhibitors

Discovery of novel antimycobacterial compounds that work on distinctive targets and by diverse mechanisms of action is urgently required for the treatment of mycobacterial infections due to the emerging global health threat of tuberculosis. We have identified a new class of 5-ethyl or hydroxy (or methoxy) methyl-substituted pyrimidine nucleosides as potent inhibitors of Mycobacterium bovis, Mycobacterium tuberculosis (H37Ra, H37Rv) and Mycobacterium avium. A series of 2'-'up' fluoro (or hydroxy) nucleosides (1, 2, 4-6, 9, 10, 13, 16, 18, 21, 24) was synthesized and evaluated for antimycobacterial activity. Among 2'-fluorinated compounds, 1-(3-bromo-2,3-dideoxy-2-fluoro-β-d-arabinofuranosyl)-5-ethyluracil (13) exhibited promising activity against M. bovis and Mtb alone, and showed synergism when combined with isoniazid. The most active compound emerging from these studies, 1-(β-d-arabinofuranosyl)-4-thio-5-hydroxymethyluracil (21) inhibited Mtb (H37Ra) (MIC(50)=0.5 μg/mL) and M. bovis (MIC(50)=0.5 μg/mL) at low concentrations, and was ten times more potent against Mtb (H37Ra) than cycloserine (MIC(50)=5.0 μg/mL), a second line drug. It also showed an additive effect when combined with isoniazid. Compound 21 retained sensitivity against a rifampicin-resistant (H37Rv) strain of Mtb (MIC(50)=1 μg/mL) at concentrations similar to that for a rifampicin-sensitive (H37Rv) strain, suggesting that it has no cross-resistance to a first-line anti-TB drug. In addition, the replication of M. avium was also inhibited by 21 (MIC(50)=10 μg/mL). No cellular toxicity of 13 or 21 was observed up to the highest concentration tested (CC(50)>100 μg/mL). These observations offer promise for a new drug treatment regimen to augment and complement the current chemotherapy of TB.     

Mycobacterium tuberculosis Infection Interferes with HIV Vaccination in Mice

Tuberculosis (TB) has emerged as the most prominent bacterial disease found in human immunodeficiency virus (HIV)-positive individuals worldwide. Due to high prevalence of asymptomatic Mycobacterium tuberculosis (Mtb) infections, the future HIV vaccine in areas highly endemic for TB will often be administrated to individuals with an ongoing Mtb infection. The impact of concurrent Mtb infection on the immunogenicity of a HIV vaccine candidate, MultiHIV DNA/protein, was investigated in mice. We found that, depending on the vaccination route, mice infected with Mtb before the administration of the HIV vaccine showed impairment in both the magnitude and the quality of antibody and T cell responses to the vaccine components p24Gag and gp160Env. Mice infected with Mtb prior to intranasal HIV vaccination exhibited reduced p24Gag-specific serum IgG and IgA, and suppressed gp160Env-specific serum IgG as compared to respective titers in uninfected HIV-vaccinated controls. Importantly, in Mtb-infected mice that were HIV-vaccinated by the intramuscular route the virus neutralizing activity in serum was significantly decreased, relative to uninfected counterparts. In addition mice concurrently infected with Mtb had fewer p24Gag-specific IFN-γ-expressing T cells and multifunctional T cells in their spleens. These results suggest that Mtb infection might interfere with the outcome of prospective HIV vaccination in humans.     

Simultaneous analysis of multiple Mycobacterium tuberculosis knockdown mutants in vitro and in vivo

Mycobacterium tuberculosis (Mtb) represents one of the most persistent bacterial threats to human health and new drugs are needed to limit its impact. Conditional knockdown mutants can help validate new drug targets, but the analysis of individual mutants is laborious and time consuming. Here, we describe quantitative DNA tags (qTags) and their use to simultaneously analyze conditional Mtb knockdown mutants that allowed silencing the glyoxylate and methylcitrate cycles (via depletion of isocitrate lyase, ICL), the serine protease Rv3671c, and the core subunits of the mycobacterial proteasome, PrcB and PrcA. The impact of gene silencing in multi-strain cultures was determined by measuring the relative abundance of mutant-specific qTags with real-time PCR. This achieved accurate quantification over a broad range of qTag abundances and depletion of ICL, Rv3671c, or PrcBA resulted in the expected impairment of growth of Mtb with butyrate as the primary carbon source, survival during oxidative stress, acid stress and starvation. The impact of depleting ICL, Rv3671c, or PrcBA in multi-strain mouse infections was analyzed with two approaches. We first measured the relative abundance of mutant-specific qTags in total chromosomal DNA isolated from bacteria that were recovered from infected lungs on agar plates. We then developed a two-step amplification procedure, which allowed us to measure the abundances of individual mutants directly in infected lung tissue. Both strategies confirmed that inactivation of Rv3671c and PrcBA severely reduced persistence of Mtb in mice. The multi-strain infections furthermore suggested that silencing ICL not only prevented growth of Mtb during acute infections but also prevented survival of Mtb during chronic infections. Analyses of the ICL knockdown mutant in single-strain infections confirmed this and demonstrated that silencing of ICL during chronic infections impaired persistence of Mtb to the extent that the pathogen was cleared from the lungs of most mice.     

The Delta fbpA mutant derived from Mycobacterium tuberculosis H37Rv has an enhanced susceptibility to intracellular antimicrobial oxidative mechanisms, undergoes limited phagosome maturation and activates macrophages and dendritic cells

Mycobacterium tuberculosis H37Rv (Mtb) excludes phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) while preventing lysosomal fusion in macrophages (MPhis). The antigen 85A deficient (Delta fbpA) mutant of Mtb was vaccinogenic in mice and the mechanisms of attenuation were compared with MPhis infected with H37Rv and BCG. Delta fbpA contained reduced amounts of trehalose 6, 6, dimycolate and induced minimal levels of SOCS-1 in MPhis. Blockade of oxidants enhanced the growth of Delta fbpA in MPhis that correlated with increased colocalization with phox and iNOS. Green fluorescent protein-expressing strains within MPhis or purified phagosomes were analysed for endosomal traffick with immunofluorescence and Western blot. Delta fbpA phagosomes were enriched for rab5, rab11, LAMP-1 and Hck suggesting enhanced fusion with early, recycling and late endosomes in MPhis compared with BCG or H37Rv. Delta fbpA phagosomes were thus more mature than H37Rv or BCG although, they failed to acquire rab7 and CD63 preventing lysosomal fusion. Finally, Delta fbpA infected MPhis and dendritic cells (DCs) showed an enhanced MHC-II and CD1d expression and primed immune T cells to release more IFN-gamma compared with those infected with BCG and H37Rv. Delta fbpA was thus more immunogenic in MPhis and DCs because of an enhanced susceptibility to oxidants and increased maturation.     

Differential regulation of protein kinase C isoforms of macrophages by pathogenic and non-pathogenic mycobacteria

Given the fact that Mycobacterium tuberculosis (Mtb) may respond to the intracellular milieu of the macrophage with the induction of environmentally regulated genes required for survival and growth of the bacteria we assumed that the protein kinases may also be the factors in Mycobacterium-macrophage interaction. Since, protein kinases play a major role in various critical cellular processes including regulation of immune responses, we describe the fate of expression and phosphorylation of protein kinase C in macrophage cell lines exposed to Mtb H37Rv and raised the question whether the change in the events of expression and phosphorylation are the results of direct interaction of bacilli with macrophages and/or, are also indirectly mediated by specific cytokines that are induced in response to exposure. Our results show that only novel PKCs are phosphorylated during infection of macrophages by pathogenic and non-pathogenic mycobacteria and the alteration is a result of direct host-bacilli association which is independent of cytokines as mediators. Expression of PKC-alpha (conventional PKC isoform) was down regulated by Mtb H37Rv. In contrast the non-pathogenic fast grower Mycobacterium smegmatis (MS) increased the expression and phosphorylation of PKC-alpha. PKC-alpha was also increased in macrophages treated with serum of mice immunized with Mtb H37Rv. The study has shown that pathogenic and non-pathogenic mycobacteria categorically select the type of protein kinases C for activation/deactivation.