Identification and characterization of a novel PrfA-regulated gene in Listeria monocytogenes whose product, IrpA, is highly homologous to internalin proteins, which contain leucine-rich repeats.
The expression of all virulence factors in Listeria monocytogenes characterized to date is controlled by the virulence regulator protein, PrfA. To identify further PrfA-regulated proteins, we examined supernatants of L. monocytogenes EGD harboring additional copies of the PrfA regulator for the presence of novel proteins. This led to the identification and biochemical purification of a hitherto uncharacterized PrfA-dependent 30-kDa protein (A. Lingnau, T. Chakraborty, K. Niebuhr, E. Domann, and J. Wehland, Infect. Immun. 64:1002-1006, 1996). Oligonucleotide primers derived from internal peptide sequences of this protein allowed the cloning and determination of the entire sequence of the respective gene. The protein comprised 297 amino acids with strong overall homology to the internalins, InlA and InlB, particularly in the region harboring the leucine-rich repeats. The gene has been designated irpA for internalin-related protein A gene. Transcriptional studies revealed that the gene was monocistronic and, like the inlA and inlB genes, was transcribed by PrfA-dependent and PrfA-independent mechanisms. Monoclonal antibodies raised against IrpA indicated that it was produced by L. monocytogenes but not by the nonpathogenic species Listeria innocua. To examine the role of IrpA in pathogenesis, we constructed an isogenic in-frame deletion mutant that removed all but 116 amino acids of the IrpA protein. This mutant was neither defective for invasion into many tissue culture cell lines nor did it demonstrate reduced intracellular survival. However, in vivo studies using the mouse infection model revealed that the irpA mutant showed reduced virulence compared to the parental strain. These results suggest a role for IrpA during disseminated infection by L. monocytogenes.
Domann
E
E
Institut für Medizinische Mikrobiologie, Justus-Liebig-Universität Giessen, Germany. eugen.domann@mikrobio.med.uni-giessen.de
Zechel
S
S
Lingnau
A
A
Hain
T
T
Darji
A
A
Nichterlein
T
T
Wehland
J
J
Chakraborty
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D018938:Caco-2 Cells; D003001:Cloning, Molecular; D004848:Epithelium / Q000382:microbiology; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006801:Humans; D008089:Listeria monocytogenes / Q000235:genetics* / Q000472:pathogenicity; D008099:Liver / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D010454:Peptide Termination Factors; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012703:Serotyping; D013696:Temperature; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D014774:Virulence / Q000235:genetics
D001426:Bacterial Proteins; D010454:Peptide Termination Factors; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015534:Trans-Activators; C069761:internalin protein, Bacteria