The Cyclic AMP Receptor Protein, CRP, Is Required for Both Virulence and Expression of the Minimal CRP Regulon in Yersinia pestis Biovar microtus

Abstract

The cyclic AMP receptor protein (CRP) is a bacterial regulator that controls more than 100 promoters, including those involved in catabolite repression. In the present study, a null deletion of the crp gene was constructed for Yersinia pestis bv. microtus strain 201. Microarray expression analysis disclosed that at least 6% of Y. pestis genes were affected by this mutation. Further reverse transcription-PCR and electrophoretic mobility shift assay analyses disclosed a set of 37 genes or putative operons to be the direct targets of CRP, and thus they constitute the minimal CRP regulon in Y. pestis. Subsequent primer extension and DNase I footprinting assays mapped transcriptional start sites, core promoter elements, and CRP binding sites within the DNA regions upstream of pla and pst, revealing positive and direct control of these two laterally acquired plasmid genes by CRP. The crp disruption affected both in vitro and in vivo growth of the mutant and led to a >15,000-fold loss of virulence after subcutaneous infection but a <40-fold increase in the 50% lethal dose by intravenous inoculation. Therefore, CRP is required for the virulence of Y. pestis and, particularly, is more important for infection by subcutaneous inoculation. It can further be concluded that the reduced in vivo growth phenotype of the crp mutant should contribute, at least partially, to its attenuation of virulence by both routes of infection. Consistent with a previous study of Y. pestis bv. medievalis, lacZ reporter fusion analysis indicated that the crp deletion resulted in the almost absolute loss of pla promoter activity. The plasminogen activator encoded by pla was previously shown to specifically promote Y. pestis dissemination from peripheral infection routes (subcutaneous infection [flea bite] or inhalation). The above evidence supports the notion that in addition to the reduced in vivo growth phenotype, the defect of pla expression in the crp mutant will greatly contribute to the huge loss of virulence of this mutant strain in subcutaneous infection.

Yersinia pestiswith Regulated Delayed Attenuation as a Vaccine Candidate To Induce Protective Immunity against Plague

“…With this goal in mind, we constructed and evaluated an araC P BAD crp strain and, for comparison, an isogenic ⌬crp strain. Our results indicate that crp affects virulence in Y. pestis, consistent with what was found in a recent study in which a Y. pestis ⌬crp strain was shown to be highly attenuated when administered subcutaneously to mice (54). In that study, the authors found that the s.c. LD 50 in mice was approximately 1 ϫ 10 4 -fold higher than that of wild-type Y. pestis strain 201 (54).…”

“…Crp modulates expression of genes in many pathogenic bacteria, including Salmonella, Vibrio cholerae, Yersinia enterocolitica, and Y. pestis (10,38,44,54). Crp regulation occurs either directly, by binding to specific DNA sequences near the regulated gene, or indirectly, through the action of CyaR, a regulatory RNA (13).…”

“…Strains of Salmonella and Y. enterocolitica with crp deletions are attenuated in mice and stimulate protective immunity against subsequent challenge with the wildtype parent (10,38). In Y. pestis, the crp gene directly regulates expression of some 37 genes, including ypkA and yopO, which encode a secreted virulence factor, and the laterally acquired plasmid genes pla, a known virulence factor, and pst, encoding the bacteriocin pesticin (38,54). In a recent report, yopJ, a secreted effector protein involved in immune suppression, was also shown to be negatively regulated by crp (55).…”

“…In a recent report, yopJ, a secreted effector protein involved in immune suppression, was also shown to be negatively regulated by crp (55). Crp is required for expression of the Ysc type 3 secretion system and other virulence factors in Yersinia, and functional loss of crp diminishes Yop secretion by Y. enterocolitica and Y. pestis (38,54). The effect of a crp mutation on LcrV secretion has not been reported.…”

“…The effect of a crp mutation on LcrV secretion has not been reported. A Y. pestis crp mutant is attenuated for virulence (54).…”