Mycobacterium tuberculosis and the closely related organism Mycobacterium bovis can survive and replicate inside macrophages. Intracellular survival is at least in part attributed to the failure of mycobacterial phagosomes to undergo fusion with lysosomes. The transformation of phagosomes into phagolysosomes involves gradual acquisition of markers from the endosomal compartment. Members of the rab family of small GTPases which confer fusion competence in the endocytic pathway are exchanged sequentially onto the phagosomal membranes in the course of their maturation. To identify the step at which the fusion capability of phagosomes containing mycobacteria is compromised, we purified green fluorescent protein-labeled M. bovis BCG phagosomal compartments (MPC) and compared GTPbinding protein profiles of these vesicles with latex bead phagosomal compartments (LBC). We report that the MPC do not acquire rab7, specific for late endosomes, even 7 days postinfection, whereas this GTP-binding protein is present on the LBC within hours after phagocytosis. By contrast, rab5 is retained and enriched with time on the MPC, suggesting fusion competence with an early endosomal compartment. Prior infection of macrophages with M. bovis BCG also affected the dynamics of rab5 and rab7 acquisition by subsequently formed LBC. Selective exclusion of rab7, coupled with the retention of rab5 on the mycobacterial phagosome, may allow organisms from the M. tuberculosis complex to avert the usual physiological destination of phagocytosed material.
Overproduction of the exopolysaccharide alginate causes mucoid colony morphology in Pseudomonas aeruginosa and is considered a major virulence determinant expressed by this organism during chronic respiratory infections in cystic fibrosis. One of the principal regulatory elements governing conversion to mucoidy in P. aeruginosa is AlgU, an alternative sigma factor which is 66% identical to and functionally interchangeable with E from Escherichia coli and Salmonella typhimurium. E has been implicated in the expression of systems enhancing bacterial resistance to environmental stress. In this study, we report that the gene encoding AlgU is transcribed in wild-type nonmucoid P. aeruginosa from multiple promoters (P 1 through P 5 ) that fall into three categories: (i) the P 1 and P 3 promoters, which display strong similarity to the ؊35 and ؊10 canonical sequences of E promoters and were found to be absolutely dependent on AlgU; (ii) the P 2 promoter, which was less active in algU mutants, but transcription of which was not completely abrogated in algU::Tc r cells; and (iii) the transcripts corresponding to P 4 and P 5 , which were not affected by inactivation of algU. Introduction of E. coli rpoE (encoding E ) or algU into P. aeruginosa algU::Tc r strains restored P 1 and P 3 transcription and brought the P 2 signal back to the wild-type level. The AlgU-dependent promoters P 1 and P 3 were inducible by heat shock in wild-type nonmucoid P. aeruginosa PAO1. At the protein level, induction of AlgU synthesis under conditions of extreme heat shock was detected by metabolic labeling of newly synthesized proteins, twodimensional gel analysis, and reaction with polyclonal antibodies raised against an AlgU peptide. Another AlgU-dependent promoter, the proximal promoter of algR, was also found to be induced by heat shock. Under conditions of high osmolarity, growth at elevated temperature induced alginate synthesis in the wild-type nonmucoid P. aeruginosa PAO1. Cumulatively, these results suggest that algU itself is subject to complex regulation and is inducible by extreme heat shock, that the alginate system is a subset of the stress-responsive elements controlled by AlgU, and that AlgU and, by extension, its homologs in other organisms (e.g., E in S. typhimurium) may play a role in bacterial virulence and adjustments to adverse growth conditions. Conversion of Pseudomonas aeruginosa to the mucoid phenotype in cystic fibrosis (CF) has become a paradigm of bacterial adaptation mechanisms in opportunistic pathogenesis (12,20,23). Mucoidy is the result of overproduction of the exopolysaccharide alginate and is considered a major virulence factor expressed by P. aeruginosa during chronic colonization of the lung in CF patients. The alginate biosynthetic pathway has been elucidated for the most part (6, 39). One of the critical alginate biosynthetic genes is algD, which encodes GDP mannose dehydrogenase, an enzyme catalyzing the first committed step in alginate synthesis (8). Transcriptional activation of this gene is necessary ...
Activation ofalgD by AlgR is essential for mucoidy, a virulence factor expressed by Pseudomonas aeruginosa in cystic fibrosis. Two AlgR-binding sites, RB1 and RB2, located far upstream from the algD mRNA start site, are essential for the high-level activity of algD. However, the removal of RB1 and RB2 does not completely
Strong transcriptional activation of algD, a key event in the overproduction of alginate and establishment of mucoidy in Pseudomonas aeruginosa, depends on the functional algR gene. The predicted gene product of algR shows homologies to response regulators from bacterial signal transduction systems. The algR gene was overexpressed in Escherichia coli, its product (AlgR) was purified by utilizing its apparent affinity for heparin, and its sequence was verified by partial amino acid sequence analysis. AlgR was found to interact directly with the algD promoter. Deletion mapping analysis, in conjunction with mobility shift DNA-binding assays, indicated the presence of three regions within the algD promoter capable of specifically binding AlgR. A relatively weak interaction was observed with the algD promoter fragment containing the region immediately upstream of the algD mRNA start site (-144 to + 11). However, when fragments spanning regions located very far upstream from the algD mRNA initiation site (-533 and -332) were used, strong specific binding was observed. These regions were separated by a DNA segment not binding AlgR and spanning positions -332 to -144. DNase I footprinting analysis further established the presence of discrete AlgR binding sites overlapping with FUS, the far-upstream sites required for full induction of algD transcription and its environmental modulation. There were two distinct binding sites: RB1, spanning nucleotides -479 to -457, and RB2, spanning nucleotides -400 to -380. Both of these sequences shared a highly conserved core region, ACCGTTCGTC. These results established a direct interaction of AlgR with the algD promoter and revealed an arrangement of binding sites highly unusual for response regulators of the AlgR type.
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