Targeting Listeria monocytogenes consensus sequence of internalin genes using an antisense molecule

The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin-based motility, the percentage of moving bacteria, and the localization of vasodilator-stimulated phosphoprotein and profilin.

The Journal of Cell Biology ◽

10.1083/jcb.135.3.647 ◽

1996 ◽

Vol 135 (3) ◽

pp. 647-660 ◽

Cited By ~ 150

Author(s):

G A Smith ◽

J A Theriot ◽

D A Portnoy

Keyword(s):

Listeria Monocytogenes ◽

Actin Polymerization ◽

Consensus Sequence ◽

Wild Type ◽

Movement Rate ◽

Bacterial Surface ◽

Rate Dependent ◽

Repeat Domain ◽

Low Levels ◽

Rate Of Movement

The ActA protein is responsible for the actin-based movement of Listeria monocytogenes in the cytosol of eukaryotic cells. Analysis of mutants in which we varied the number of proline-rich repeats (PRR; consensus sequence DFPPPPTDEEL) revealed a linear relationship between the number of PRRs and the rate of movement, with each repeat contributing approximately 2-3 microns/min. Mutants lacking all functional PRRs (generated by deletion or point mutation) moved at rates 30% of wild-type. Indirect immunofluorescence indicated that the PRRs were directly responsible for binding of vasodilator-stimulated phosphoprotein (VASP) and for the localization of profilin at the bacterial surface. The long repeats, which are interdigitated between the PRRs, increased the frequency with which actin-based motility occurred by a mechanism independent of the PRRs, VASP, and profilin. Lastly, a mutant which expressed low levels of ActA exhibited a phenotype indicative of a threshold; there was a very low percentage of moving bacteria, but when movement did occur, it was at wild-type rates. These results indicate that the ActA protein directs at least three separable events: (1) initiation of actin polymerization that is independent of the repeat region; (2) initiation of movement dependent on the long repeats and the amount of ActA; and (3) movement rate dependent on the PRRs.

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Listeria monocytogenes σB Regulates Stress Response and Virulence Functions

Journal of Bacteriology ◽

10.1128/jb.185.19.5722-5734.2003 ◽

2003 ◽

Vol 185 (19) ◽

pp. 5722-5734 ◽

Cited By ~ 246

Author(s):

Mark J. Kazmierczak ◽

Sharon C. Mithoe ◽

Kathryn J. Boor ◽

Martin Wiedmann

Keyword(s):

Gene Expression ◽

Listeria Monocytogenes ◽

Stress Response ◽

Consensus Sequence ◽

Virulence Gene ◽

Wild Type ◽

Promoter Regions ◽

Virulence Gene Expression ◽

Stress Response Genes ◽

In The Wild

ABSTRACT While the stress-responsive alternative sigma factor σB has been identified in different species of Bacillus, Listeria, and Staphylococcus, theσ B regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify σB-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidateσ B-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted σB-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and ΔsigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significantσ B-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting σB-dependent expression, 54 were preceded by a sequence resembling the σB promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the σB-dependent nature of a subset of eight selected promoter regions. Notably, theσ B-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, σB also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest thatσ B contributes to L. monocytogenes gene expression during infection.

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LPXTG Protein InlJ, a Newly Identified Internalin Involved in Listeria monocytogenes Virulence

Infection and Immunity ◽

10.1128/iai.73.10.6912-6922.2005 ◽

2005 ◽

Vol 73 (10) ◽

pp. 6912-6922 ◽

Cited By ~ 105

Author(s):

Christophe Sabet ◽

Marc Lecuit ◽

Didier Cabanes ◽

Pascale Cossart ◽

Hélène Bierne

Keyword(s):

Listeria Monocytogenes ◽

Deletion Mutant ◽

Consensus Sequence ◽

Surface Proteins ◽

Leucine Rich Repeat ◽

Food Borne ◽

Oral Inoculation ◽

Protein Encoding ◽

Encoding Genes ◽

Lrr Protein

ABSTRACTListeria monocytogenesexpresses surface proteins covalently anchored to the peptidoglycan by sortase enzymes. Inactivation ofsrtAattenuatesListeriavirulence in mice (H. Bierne, S. K. Mazmanian, M. Trost, M. G. Pucciarelli, G. Liu, P. Dehoux, L. Jansch, F. Garcia-del Portillo, O. Schneewind, and P. Cossart, Mol. Microbiol.43:869-881, 2002). We show here that ansrtAmutant is more attenuated than an internalin mutant in orally infected guinea pigs and transgenic mice expressing human E-cadherin (hEcad mice), indicating the involvement of other SrtA substrates, LPXTG proteins, in food-borne listeriosis. Data recently generated with a listerial DNA macroarray identified two LPXTG protein-encoding genes present in the genomes ofL. monocytogenesstrains and absent from all otherListeriaspecies,inlI(lmo0333) andinlJ(lmo2821). They also revealed two other LPXTG protein-encoding genes, ORF29 and ORF2568, present only in a subclass ofL. monocytogenesserovars, including the epidemic serovar 4b. We report here that aninlJdeletion mutant, in contrast toinlIand ORF29 mutants, is significantly attenuated in virulence after intravenous infection of mice or oral inoculation of hEcad mice. Interestingly, a ΔORF2568 strain showed a slight increase in virulence.inlJencodes a leucine-rich repeat (LRR) protein that is structurally related to the listerial invasion factor internalin. However, the consensus sequence of the InlJ LRR defines a novel subfamily of cysteine-containing LRRs in bacteria. In conclusion, this postgenomic approach identified InlJ as a new virulence factor among the proteins belonging to the internalin family inL. monocytogenes.

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