The growth rate of <i>Mycobacterium smegmatis</i> depends on sufficient porin‐mediated influx of nutrients

Stephan, Joachim; Bender, Jennifer K.; Wolschendorf, Frank; Hoffmann, Christian; Roth, E.; Mailänder, Claudia; Engelhardt, Harald; Niederweis, Michael

doi:10.1111/j.1365-2958.2005.04878.x

Cited by 100 publications

(166 citation statements)

References 51 publications

(103 reference statements)

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“…Proteins were extracted from M. smegmatis by using 2% SDS in PBS at 40°C as described (13). Proteins were analyzed in Western blots by using specific antibodies raised against MctB (5D1.23), MymT (kindly provided by Ben Gold), and horseradish peroxidase-coupled anti-mouse or anti-rabbit secondary antibodies as described (30). For further details, see SI Appendix.…”

Section: Methodsmentioning

confidence: 99%

Copper resistance is essential for virulence of Mycobacterium tuberculosis

Wolschendorf

Ackart

Shrestha

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Copper (Cu) is essential for many biological processes, but is toxic when present in excessive amounts. In this study, we provide evidence that Cu plays a crucial role in controlling tuberculosis. A Mycobacterium tuberculosis ( Mtb ) mutant lacking the outer membrane channel protein Rv1698 accumulated 100-fold more Cu and was more susceptible to Cu toxicity than WT Mtb . Similar phenotypes were observed for a M. smegmatis mutant lacking the homolog Ms3747, demonstrating that these mycobacterial copper transport proteins B (MctB) are essential for Cu resistance and maintenance of low intracellular Cu levels. Guinea pigs responded to infection with Mtb by increasing the Cu concentration in lung lesions. Loss of MctB resulted in a 1,000- and 100-fold reduced bacterial burden in lungs and lymph nodes, respectively, in guinea pigs infected with Mtb . In mice, the persistence defect of the Mtb mctB mutant was exacerbated by the addition of Cu to the diet. These experiments provide evidence that Cu is used by the mammalian host to control Mtb infection and that Cu resistance mechanisms are crucial for Mtb virulence. Importantly, Mtb is much more susceptible to Cu than other bacteria and is killed in vitro by Cu concentrations lower than those found in phagosomes of macrophages. Hence, this study reveals an Achilles heel of Mtb that might be a promising target for tuberculosis chemotherapy.

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Section: Methodsmentioning

confidence: 99%

Copper resistance is essential for virulence of Mycobacterium tuberculosis

Wolschendorf

Ackart

Shrestha

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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281

333

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“…The CpnT NTD-HA-His6 protein was purified from the M. smegmatis strain ML1910, which lacks all known endogenous porin genes and conditionally expresses cpnT NTD , to avoid contamination with M. smegmatis and M. tuberculosis pores. The porin-deletion mutant ML1910 is not viable without expression of cpnT NTD , indicating that it does not have outer membrane proteins with significant channel activity other than the Msp porins (25). The CpnT NTD protein was extracted from the M. smegmatis porin-deletion mutant with SDS and purified by Ni (II)-affinity followed by anion-exchange chromatography.…”

Section: Significancementioning

confidence: 99%

An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity

Danilchanka¹,

Sun²,

Pavlenok³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The ability to control the timing and mode of host cell death plays a pivotal role in microbial infections. Many bacteria use toxins to kill host cells and evade immune responses. Such toxins are unknown in Mycobacterium tuberculosis. Virulent M. tuberculosis strains induce necrotic cell death in macrophages by an obscure molecular mechanism. Here we show that the M. tuberculosis protein Rv3903c (channel protein with necrosis-inducing toxin, CpnT) consists of an N-terminal channel domain that is used for uptake of nutrients across the outer membrane and a secreted toxic C-terminal domain. Infection experiments revealed that CpnT is required for survival and cytotoxicity of M. tuberculosis in macrophages. Furthermore, we demonstrate that the C-terminal domain of CpnT causes necrotic cell death in eukaryotic cells. Thus, CpnT has a dual function in uptake of nutrients and induction of host cell death by M. tuberculosis.transport | pore | secretion

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“…Consecutive deletions of the two porin genes mspA and mspC reduced the number of pores 15-fold compared to wild-type M. smegmatis. The loss of porins lowered the permeability for glucose 75-fold and, concomitantly, the growth rate of M. smegmatis on plates and in liquid medium dropped drastically (Stephan et al, 2005). This showed for the first time that the porinmediated influx of hydrophilic nutrients limited the growth rate of porin mutants.…”

Section: Transport Across Mycobacterial Outer Membranesmentioning

confidence: 80%

“…However, it is unknown which and how many nutrients are in low supply in the M. smegmatis porin mutants. Since MspA could not be expressed in M. smegmatis above wild-type levels (Stephan et al, 2005), it is also not clear whether the influx of nutrients really limits the growth rate of wild-type mycobacteria as suggested earlier (Jarlier & Nikaido, 1990). The fact that the lack of the MspA and MspC porins also caused a reduced uptake of phosphates and slower growth on low-phosphate plates (Wolschendorf et al, 2007) indeed suggested that the slow uptake of essential hydrophilic nutrients other than the carbon source may also contribute to the slow growth of M. smegmatis porin mutants.…”

Section: Transport Across Mycobacterial Outer Membranesmentioning

confidence: 99%

“…Note that no experimental data exist for either the spatial distribution of the extractable lipids, or the confirmations and exact locations of the mycolic acids. The porin MspA mediates the uptake of small and hydrophilic nutrients such as sugars (Stephan et al, 2005) and phosphates (Wolschendorf et al, 2007) across the outer membrane of M. smegmatis. The representation of MspA is based on its crystal structure (Faller et al, 2004) and was created using the visualization software PyMol (DeLano Scientific).…”

Section: Permeability Barriers In Mycobacterial Cell Envelopesmentioning

confidence: 99%

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Nutrient acquisition by mycobacteria

2008

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The growth and nutritional requirements of mycobacteria have been intensively studied since the discovery of Mycobacterium tuberculosis more than a century ago. However, the identity of many transporters for essential nutrients of M. tuberculosis and other mycobacteria is still unknown despite a wealth of genomic data and the availability of sophisticated genetic tools. Recently, considerable progress has been made in recognizing that two lipid permeability barriers have to be overcome in order for a nutrient molecule to reach the cytoplasm of mycobacteria. Uptake processes are discussed by comparing M. tuberculosis with Mycobacterium smegmatis. For example, M. tuberculosis has only five recognizable carbohydrate transporters in the inner membrane, while M. smegmatis has 28 such transporters at its disposal. The specificities of inner-membrane transporters for sulfate, phosphate and some amino acids have been determined. Outer-membrane channel proteins in both organisms are thought to contribute to nutrient uptake. In particular, the Msp porins have been shown to be required for uptake of carbohydrates, amino acids and phosphate by M. smegmatis. The set of porins also appears to be different for M. tuberculosis and M. smegmatis. These differences likely reflect the lifestyles of these mycobacteria and the availability of nutrients in their natural habitats: the soil and the human body. The comprehensive identification and the biochemical and structural characterization of the nutrient transporters of M. tuberculosis will not only promote our understanding of the physiology of this important human pathogen, but might also be exploited to improve tuberculosis chemotherapy. IntroductionThe growth and nutritional requirements of mycobacteria have been intensively studied since the discovery of Mycobacterium tuberculosis (Koch, 1882). These studies have resulted in an overwhelming body of literature on the physiology of mycobacterial metabolism in the years before the dawn of molecular biology (Edson, 1951;Ramakrishnan et al., 1972;Ratledge, 1982). For example, the utilization of many solutes such as carbohydrates, alcohols, carboxy acids, fatty acids and amino acids by mycobacteria was examined by measuring oxygen consumption (Edson, 1951). It is striking that the identity of many transporters for essential nutrients of M. tuberculosis and other mycobacteria is still unknown despite considerable progress in the development of genetic methods for mycobacteria (Kana & Mizrahi, 2004;Machowski et al., 2005). Recently, in particular carbon metabolism of mycobacteria has attracted renewed interest since the observation that M. tuberculosis relies on the glyoxylate cycle for survival in mice (McKinney et al., 2000; MunozElias & McKinney, 2005). This indicates that M. tuberculosis uses lipids as the main carbon source during infection. On the other hand, genes that encode a putative disaccharide transporter were found to be essential for M. tuberculosis during the first week of infection (Sassetti & Rubin, 2003), indicating...

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The growth rate of Mycobacterium smegmatis depends on sufficient porin‐mediated influx of nutrients

Cited by 100 publications

References 51 publications

Copper resistance is essential for virulence of Mycobacterium tuberculosis

Copper resistance is essential for virulence of Mycobacterium tuberculosis

An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity

Nutrient acquisition by mycobacteria

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