The NlpD Lipoprotein Is a Novel Yersinia pestis Virulence Factor Essential for the Development of Plague

Abstract

Yersinia pestis is the causative agent of plague. Previously we have isolated an attenuated Y. pestis transposon insertion mutant in which the pcm gene was disrupted. In the present study, we investigated the expression and the role of pcm locus genes in Y. pestis pathogenesis using a set of isogenic surE, pcm, nlpD and rpoS mutants of the fully virulent Kimberley53 strain. We show that in Y. pestis, nlpD expression is controlled from elements residing within the upstream genes surE and pcm. The NlpD lipoprotein is the only factor encoded from the pcm locus that is essential for Y. pestis virulence. A chromosomal deletion of the nlpD gene sequence resulted in a drastic reduction in virulence to an LD50 of at least 107 cfu for subcutaneous and airway routes of infection. The mutant was unable to colonize mouse organs following infection. The filamented morphology of the nlpD mutant indicates that NlpD is involved in cell separation; however, deletion of nlpD did not affect in vitro growth rate. Trans-complementation experiments with the Y. pestis nlpD gene restored virulence and all other phenotypic defects. Finally, we demonstrated that subcutaneous administration of the nlpD mutant could protect animals against bubonic and primary pneumonic plague. Taken together, these results demonstrate that Y. pestis NlpD is a novel virulence factor essential for the development of bubonic and pneumonic plague. Further, the nlpD mutant is superior to the EV76 prototype live vaccine strain in immunogenicity and in conferring effective protective immunity. Thus it could serve as a basis for a very potent live vaccine against bubonic and pneumonic plague.

A Novel Zinc Binding System, ZevAB, Is Critical for Survival of Nontypeable Haemophilus influenzae in a Murine Lung Infection Model

“…of 0.3 or lower and sustaining insertions in Ͼ40% of the potential TA dinucleotide insertion sites, specific to the himar1-derived transposon used in the study (22). Comparison of our list of potential DsbA substrates to the results of HITS revealed three genes that fit these criteria: znuA (s.i., 0.101), encoding the periplasmic component of a high-affinity zinc uptake transporter (39, 53), nlpD (s.i., 0.000), encoding a lipoprotein suggested to be involved in daughter cell separation (64,65), and HI1249 (s.i., 0.106), encoding a putative protein herein referred to as ZevA. The role of znuA in virulence has been studied by several groups (3,11,14,21,36,57,75), and nlpD has been implicated in the virulence of Yersinia pestis and Yersinia enterocolitica (12,64).…”

“…Here, we generated a list of potential substrates of the periplasmic disulfide oxidoreductase protein DsbA (see Table S2 in the supplemental material) and integrated our findings with the results of a previous study in which HITS technology was used to identify genes required for lung infection (22). Using this strategy, we identified three genes that were potentially important for lung infection, including two, znuA and nlpD, that had previously been implicated in the virulence of several organisms (3,11,12,14,21,36,57,64,75), and a third gene, zevA, whose function had not been determined. Therefore, zevA and its neighboring gene, zevB, were investigated for their roles in the pathogenesis of a clinical NTHI isolate.…”

Evolutionary convergence in experimental Pseudomonas populations

“…Genome-sequencing data available from that study (of line 6B 4 ) suggested candidate genes and Sanger sequencing was used to find the mutational causes. In 23 out of 24 strains a mutation was found in PFLU1301 (nlpD), which encodes a lipoprotein that in E. coli (Uehara et al, 2010), Vibrio cholerae (Moll et al, 2014) and Yersinia pestis (Tidhar et al, 2009) is an activator of AmiC (N-acetylmuramoyl-L-alanine amidase) hydrolase activity required for proper cell division: loss-offunction mutations in nlpD in these species exhibit a similar cellular morphology as observed for the CC types and can be complemented by expression in trans. In all 23 cases the mutation in nlpD was identical-a single Q189* (CAG-4TAG) nonsense mutation.…”

In silico prediction of

“…Gab_1309 is a predicted lipoprotein, and this annotation is supported by the DOLOP database [50]. The protein sequence of Gab_1309 shows sequence similarity to the NlpD lipoprotein from Yersinia pestis [51]. By use of the Conserved Domain Database (CDD) feature in BLASTp [52], an N-terminal LysM domain and a Cterminal M23 peptidase domain can be identified in Gab_1309.…”