Virulence Conferred by PumA Toxin from the Plasmid-Encoded PumAB Toxin-Antitoxin System is Regulated by Quorum System.

Toxin-antitoxin (TA) systems are small genetic elements composed of a toxin gene and its cognate antitoxin that are important for plasmid stabilization (plasmid-encoded) and bacterial virulence (chromosome-encoded). These systems are also related to biofilm and persister cell formations. Pseudomonas aeruginosa is an antibiotic-resistant human pathogen that produces virulence factors modulated by quorum sensing (QS) and can form biofilms. The type II PumAB TA system of pUM505, isolated from a clinical strain of P. aeruginosa, confers plasmid stability. Additionally, the PumA toxin increases P. aeruginosa virulence and is neutralized by the PumB antitoxin. In this study, we determined whether virulence conferred by PumA toxin is regulated by QS. The pumA gene was transferred to P. aeruginosa lasI/rhlI, a mutant strain in the LasI and RhlI QS systems, to analyze the effect on virulence of the transformants. pumA transfer did not increase bacterial virulence in lettuce and Caenorhabditis elegans, suggesting that the virulence conferred by PumA requires QS modulation. pumA mRNA levels drastically decreased in the P. aeruginosa lasI/rhlI (pUC_pumA) strain, suggesting positive regulation of pumA gene expression by QS. Supplementation of the growth medium of P. aeruginosa lasI/rhlI (pUC_pumA) with C4-AHL and 3-oxo-C12-AHL autoinducers increased pumA mRNA levels and restored bacterial virulence, suggesting that both autoinducers complemented the mutations and positively regulated the toxic effects of PumA. This strengthened the hypothesis that QS regulates bacterial virulence conferred by the PumA toxin. Thus, this report establishes an important function of QS in the virulence conferred by plasmid-encoded TA systems in bacterial pathogens.

Prevalence of the crpP gene conferring decreased ciprofloxacin susceptibility in enterobacterial clinical isolates from Mexican hospitals.

OBJECTIVES: This study investigated the presence of the crpP gene, which encodes an enzymatic mechanism of antibiotic phosphorylation that decreases ciprofloxacin susceptibility, in ESBL-producing clinical isolates and its effect in transconjugants. METHODS: A collection of 77 ESBL-producing clinical isolates of Enterobacteriaceae and 68 ESBL-producing transconjugants that had acquired plasmids from clinical isolates from hospitals in Mexico obtained from 1988 to 2012 was employed. The crpP homologue genes were identified by dot-blot and PCR assays; five of them were sequenced and an in silico analysis was conducted. Expression of CrpP proteins was determined by western blot assays using antibodies against CrpP from plasmid pUM505. Three crpP homologue genes were cloned and transferred to Escherichia coli J53-3 as recipient strain. RESULTS: The crpP gene was identified in four (5.19%) ESBL-producing isolates and five (7.35%) ESBL-producing transconjugants with plasmids from clinical isolates. Analysis of the deduced amino acid (aa) sequence of the CrpP protein homologues revealed that they all corresponded to small proteins (63-70 aa) with an identity of 10.1%-43.7% with respect to the pUM505 CrpP sequence. In addition, all crpP-positive transconjugants expressed a CrpP protein. Finally, transfer of crpP homologues conferred lower ciprofloxacin susceptibility to E. coli. CONCLUSIONS: These findings indicate the presence of crpP genes among ESBL-producing isolates from Mexican hospitals and point to widespread crpP-type genes in old Enterobacteriaceae clinical isolates (from 1994). CrpP probably confers resistance by means of the phosphorylation of ciprofloxacin.

A plasmid-encoded mobile genetic element from Pseudomonas aeruginosa that confers heavy metal resistance and virulence.

Mobile plasmid-encoded elements are DNA segments that are transferred for horizontal gene transfer and that confer adaptive proprieties, as well as virulence and antibiotic and heavy metal resistance to bacteria. The conjugative plasmid pUM505, isolated from a clinical strain of Pseudomonas aeruginosa, possesses a putative 31.292 kb mobile element (denominated Mpe: Mobile plasmid- encoded element) that, in addition to possessing chr genes that confer chromate resistance to Pseudomonas, contains two putative mer operons that could confer mercury resistance. Moreover, the Mpe contains genes related previously with the virulence of both P. aeruginosa and Escherichia coli strains. In this work, we determined that Mpe from pUM505 was able to independently move to another DNA molecule, conferring chromate and mercury resistance to P. aeruginosa PAO1 and mercury resistance to E. coli JM101, suggesting that its transference might be beneficial to bacteria under certain environmental conditions. Additionally, the transference of Mpe increased the virulence of P. aeruginosa PAO1 against the nematode Caenorhabditis elegans, suggesting its contribution to the pathogenicity of P. aeruginosa. In this work, we describe a new mobile plasmid-encoded element that possesses the potential to be transferred by horizontal gene transference, which could provide bacteria with a wide variety of adaptive traits such as heavy metal resistance and virulence, which can be selective factors for the distribution and prevalence of this plasmid in diverse environments, including hospitals and heavy metal contaminated soils.

CrpP Is a Novel Ciprofloxacin-Modifying Enzyme Encoded by the Pseudomonas aeruginosa pUM505 Plasmid.

The pUM505 plasmid, isolated from a clinical Pseudomonas aeruginosa isolate, confers resistance to ciprofloxacin (CIP) when transferred into the standard P. aeruginosa strain PAO1. CIP is an antibiotic of the quinolone family that is used to treat P. aeruginosa infections. In silico analysis, performed to identify CIP resistance genes, revealed that the 65-amino-acid product encoded by the orf131 gene in pUM505 displays 40% amino acid identity to the Mycobacterium smegmatis aminoglycoside phosphotransferase (an enzyme that phosphorylates and inactivates aminoglycoside antibiotics). We cloned orf131 (renamed crpP, for ciprofloxacin resistance protein, plasmid encoded) into the pUCP20 shuttle vector. The resulting recombinant plasmid, pUC-crpP, conferred resistance to CIP on Escherichia coli strain J53-3, suggesting that this gene encodes a protein involved in CIP resistance. Using coupled enzymatic analysis, we determined that the activity of CrpP on CIP is ATP dependent, while little activity against norfloxacin was detected, suggesting that CIP may undergo phosphorylation. Using a recombinant His-tagged CrpP protein and liquid chromatography-tandem mass spectrometry, we also showed that CIP was phosphorylated prior to its degradation. Thus, our findings demonstrate that CrpP, encoded on the pUM505 plasmid, represents a new mechanism of CIP resistance in P. aeruginosa, which involves phosphorylation of the antibiotic.