An increase in expression of a Mycobacterium tuberculosis mycolyl transferase gene (fbpB) occurs early after infection of human monocytes

Abstract

SummaryChanges in the mRNA levels of two Mycobacterium tuberculosis genes (fbpB known as antigen 85B, and hspX known as Acr) were studied in infected human monocytes. Antigen 85B is an enzyme involved in cell wall biosynthesis and is also a major target of the immune response. Acr is a stress protein believed to be involved in the bacillary response to adverse conditions and in non-replicating persistence. During the first 24 h of intracellular infection, the intramonocyte 85B mRNA level increased 54-fold (P 0.00001) and 14.6 times in comparison with the 16S ribosomal rRNA. In contrast, the Acr mRNA fell 14.3 times. Although monocyte cytokine production was very variable, the 24 h secretion of tumour necrosis factor (TNF)-a correlated with the 85B216S RNA ratio at 24 h (r 0.77, P corr , 0.01). Furthermore, the addition of exogenous TNF-a to cultures was associated with a twofold increase in the 85B216S ratio and, conversely, neutralization of endogenous TNF-a reduced the ratio. As antigen 85B also induces TNFa, the positive feedback implied by our findings suggests a previously unsuspected role for this protein in the immunopathogenesis of tuberculosis.

A deletion defining a common Asian lineage ofMycobacterium tuberculosisassociates with immune subversion

“…The extraction of MTB RNA from strains grown in 7H9 broth was performed as described in ref. 25. Reverse transcription was carried out for 1 h at 37°C by using 0.1 g͞ l random primers (Promega), 0.28 unit͞ l AMV-RT (ABgene, Epsom, U.K.) in the supplier's reaction buffer, and a 500 M concentration of each dNTP (Qiagen).…”

Mycobacterium tuberculosis Origin of Replication and the Promoter for Immunodominant Secreted Antigen 85B Are the Targets of MtrA, the Essential Response Regulator

“…These proteins catalyze mycolyl transferase activity (18), which is critical for generating ␣-␣-trehalose dimycolate or "cord factor", a major virulence factor (19,20). Also, infection of monocyte-derived macrophages with Mtb is shown to be associated with a sharp increase in the expression of fbpB, and such a rapid increase is believed to be necessary for establishing infection and subsequent proliferation in macrophages (21). The expression of fbpA is differentially modulated during the acute and chronic stages of infection (22)(23)(24).…”

Suppression of Mycobacterium tuberculosis induced reactive oxygen species (ROS) and TNF‐α mRNA expression in human monocytes by allicin

“…Blood was collected by venipuncture in heparinized syringes and PBMC (peripheral blood mononuclear cells) were isolated by density gradient sedimentation on Ficoll-Paque separation medium [14]. PBMC (5 · 10 6 cells/well) were added in 12-well tissue culture plates (Costar Corp., Cambridge, MA) in complete RPMI-1640 medium, and were subsequently incubated at 37°C, 5% CO 2 for 1-2 h for adherence.…”

“…Mid-log cultures of MTB (H 37 Rv) grown in Middlebrook 7H9 broth (HiMedia, India) and quantified by CFU assay were used for infection [14,15]. Adherant monocytes (MNs) were infected with MTB at 1:1 (bacteria/cell) in 30% autologous serum for 90 min at 37°C, 5% CO 2 , followed by washing (4·) with RPMI-1640 medium.…”

“…Human R18 housekeeping gene was employed to normalize gene expression. TaqManä PCR primers and probes as well as target-specific RT primer for each assay were designed as described elsewhere [14,17].…”