Identification of Virulence Genes in a Pathogenic Strain of Pseudomonas aeruginosa by Representational Difference Analysis

Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
Journal of Bacteriology (Impact Factor: 2.81). 03/2002; 184(4):952-61. DOI: 10.1128/jb.184.4.952-961.2002
Source: PubMed


Pseudomonas aeruginosa is an opportunistic pathogen that may cause severe infections in humans and other vertebrates. In addition, a human clinical
isolate of P. aeruginosa, strain PA14, also causes disease in a variety of nonvertebrate hosts, including plants, Caenorhabditis elegans, and the greater wax moth, Galleria mellonella. This has led to the development of a multihost pathogenesis system in which plants, nematodes, and insects have been used
as adjuncts to animal models for the identification of P. aeruginosa virulence factors. Another approach to identifying virulence genes in bacteria is to take advantage of the natural differences
in pathogenicity between isolates of the same species and to use a subtractive hybridization technique to recover relevant
genomic differences. The sequenced strain of P. aeruginosa, strain PAO1, has substantial differences in virulence from strain PA14 in several of the multihost models of pathogenicity,
and we have utilized the technique of representational difference analysis (RDA) to directly identify genomic differences
between P. aeruginosa strains PA14 and PAO1. We have found that the pilC, pilA, and uvrD genes in strain PA14 differ substantially from their counterparts in strain PAO1. In addition, we have recovered a gene homologous
to the ybtQ gene from Yersinia, which is specifically present in strain PA14 but absent in strain PAO1. Mutation of the ybtQ homolog in P. aeruginosa strain PA14 significantly attenuates the virulence of this strain in both G. mellonella and a burned mouse model of sepsis to levels comparable to those seen with PAO1. This suggests that the increased virulence
of P. aeruginosa strain PA14 compared to PAO1 may relate to specific genomic differences identifiable by RDA.


Available from: Laurence G Rahme
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    • "G. mellonella larvae have been shown as a validated invertebrate host model for deciphering virulence in K. pneumoniae[11,12] as well as a number of other bacterial pathogens including: Listeria spp. [29], Staphylococcus aureus[16], Acinetobacter baumannii[15], Pseudomonas aeruginosa[30], among others. Several studies have also used the G. mellonella model to demonstrate the virulence between different strains or genetic mutations of organisms [11,15,16]. "
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    ABSTRACT: Previous studies may have overestimated morbidity and mortality due to Klebsiella pneumoniae producing carbapenemase (KPC) Klebsiella pneumoniae infections because of difficulties in modeling patient comorbidities. This pilot study sought to evaluate KPC virulence by combining clinical and Galleria mellonella models in patients with K. pneumoniae blood stream infections (BSIs). G. mellonella were inoculated using KPC(+) and KPC(-) isolates from these patients. Extent and rapidity of insect mortality was analyzed. Patients were stratified by KPC BSI status. Clinical outcomes of mortality and length of stay post-infection for survivors (LOS) were analyzed. Median virulence scores calculated from the insect studies were imputed in the clinical model. The in-vivo model revealed greater mortality in KPC(-) isolates (p < 0.001). Fifteen patients with KPC(+) BSI were matched with 60 patients with KPC(-) BSI. Hospital mortality was greater in the KPC(+) group versus the KPC(-) group (OR 3.79, 95% CI 1.00 - 14.34). LOS was longer in the KPC(+) group (p < 0.01). Conversely the virulence score attenuated the association between KPC(+) status and mortality and LOS in the final translational models. KPC(+) status was associated with decreased virulence in GM. Opposite findings were observed in patients. This pilot study demonstrates that virulence from GM and humans may differ.
    BMC Infectious Diseases 01/2014; 14(1):31. DOI:10.1186/1471-2334-14-31 · 2.61 Impact Factor
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    • "Here, we observed that the initial production phase of HCN/CN − by strain PA14 was more rapid than by strain MPAO1 which is widely used as a reference strain for studying P. aeruginosa PAO1 virulence (Fig. 3B). Clinical isolate PA14 is significantly more virulent than PAO1 in a wide range of hosts, including mice, nematodes, insects as Galleria mellonella, and plants as Arabidopsis thaliana (Choi et al., 2002; Rahme et al., 1995; Tan et al., 1999). Concerning , the well characterized host infection model, C. elegans, it has been shown that strain PA14 is able to kill these worms more efficiently than PAO1 (Tan et al., 1999 "
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    ABSTRACT: Pseudomonas aeruginosa is an opportunistic pathogen responsible for numerous infections acquired in hospital especially in persons whose immune systems are weakened, such as with patient suffering from AIDS or cystic fibrosis. This bacterium produces a great diversity of virulence factors among them hydrogen cyanide (HCN) which is one of the most potent and toxic. A precise quantification of HCN or CN(-) ion is essential to understand the involvement of this toxin in the pathogenesis of P. aeruginosa. In the present study, we present a new technique based on a polarographic approach to measure the production kinetics of HCN/CN(-) by P. aeruginosa strains, in several media commonly used in microbiology labs. The method was validated using mutants (hcnB- and hcnC-) which are unable to produce detectable HCN/CN(-). The kinetics of HCN/CN(-) production by P. aeruginosa in Luria Bertani (LB) medium showed a parabolic shape with a peak observed at 4, 5 and 8h for strains PA14, PAO1 and MPAO1, respectively. When bacteria were grown in ordinary nutrient broth (ONB) 2.5% medium, a less adapted medium for bacterial growth, the general profile of the kinetics was conserved but peak production was delayed (10 and 12h for PAO1 and MPAO1, respectively). When the bacteria were cultured in minimum medium MMC, bacterial growth was particularly slow and HCN/CN(-) production was markedly reduced. Taken together, this new polarographic method appears as a useful technique to detect and quantify HCN/CN(-) in routine media where the bacteria can express and regulate high amounts of toxins. With this method, we demonstrate that HCN/CN(-) production by P. aeruginosa is maximal at the end of the exponential growth phase and depends on the richness of the growth medium used.
    Journal of microbiological methods 04/2012; 90(1):20-4. DOI:10.1016/j.mimet.2012.04.005 · 2.03 Impact Factor
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    • "In addition to mammalian systems, the models include plants (lettuce leaves and Arabidopsis thaliana), invertebrates (Caenorhabditis elegans), insects (Galleria mellonella and Drosophila melanogaster) and vertebrates (zebrafish) [11], [12], [13]. Although PAO1 is infectious in some of these models, the majority of them employ PA14, which displays higher virulence in most hosts [5], [14], [15]. "
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    ABSTRACT: Pseudomonas aeruginosa is a pathogen that causes acute and chronic infections in a variety of hosts. The pathogenic potential of P. aeruginosa is strain-dependent. PA14 is a highly virulent strain that causes disease in a wide range of organisms, whereas PAO1 is moderately virulent. Although PA14 carries pathogenicity islands that are absent in PAO1, the presence or absence of specific gene clusters is not predictive of virulence. Here, we show that the virulent strain PA14 has an acquired mutation in the ladS gene. This mutation has a deleterious impact on biofilm, while it results in elevated type III secretion system (T3SS) activity and increased cytotoxicity towards mammalian cells. These phenotypes can be reverted by repairing the ladS mutation on the PA14 genome. The RetS/LadS/GacS signaling cascade is associated with virulence and the switch between acute and chronic infections. RetS is a sensor that down-regulates biofilm formation and up-regulates the T3SS. Mutations in retS are acquired in strains isolated from chronically infected cystic fibrosis patients and lead to hyperbiofilm formation and reduced cytotoxicity. Conversely, the LadS sensor promotes biofilm formation and represses the T3SS. We conclude that the ladS mutation is partly responsible for the high cytotoxicity of PA14, and our findings corroborate the central role of RetS and LadS in the switch between acute and chronic infections. Given the extensive use of the reference strain PA14 in infection and virulence models, the bias caused by the ladS mutation on the observed phenotypes will be crucial to consider in future research.
    PLoS ONE 12/2011; 6(12):e29113. DOI:10.1371/journal.pone.0029113 · 3.23 Impact Factor
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