Impact of Peptidoglycan Recycling Blockade and Expression of Horizontally Acquired β-Lactamases on Pseudomonas aeruginosa Virulence

Abstract

Pseudomonas aeruginosa is one of the leading nosocomial pathogens whose growing resistance makes the development of therapeutic options extremely urgent. The resistance-virulence interplay has classically aroused researchers’ interest as a source of therapeutic targets.

Role of Enzymatic Activity in the Biological Cost Associated with the Production of AmpC β-Lactamases in Pseudomonas aeruginosa

“…Regarding AmpC/AmpC* production from pUCP24-derived plasmids, the motility results are not as clear as those in the triple-amidase-defective background, and although some similar trends can be appreciated, they either did not reach or showed only weak statistical significance. In any case, these results are in accordance with our previous work ( 25 ) in which motility was demonstrated to be barely altered in the PAΔAG strain expressing different OXA-2-type enzymes despite the existence of large differences between OXA-2-type enzyme-associated virulence behaviors in G. mellonella .…”

“…This circumstance suggests the existence of particularities in the output and probably the mechanistic basis for the cost linked to each of the two paths used to achieve AmpC hyperproduction and recycling impairment, i.e., triple-amidase deficiency versus AmpG disruption plus pUCPAC expression. In any case, the lack of significant handicaps for the exponential growth and cytotoxicity of PA14ΔAG/pUCPAC compared to the appropriate controls (PA14ΔAG/pUCP24, for instance) is in accordance with previous results regarding the production of pUCP24-cloned OXA-2-like enzymes in the same background, which involved very important alterations in virulence in G. mellonella without affecting cytotoxicity and duplication times ( 25 ).…”

“…A similar trend, although much less pronounced, was seen when AmpC and AmpC* were produced from their respective genes located in pUCP24-derived plasmids: whereas the expression of pUCPAC in PAO1 entailed a statistically significant approximate duplication of the LD 50 value, wild-type behavior was restored in the strain expressing pUCPAC*, which showed virtually the same LD 50 as that of PAO1/pUCP24. As recently demonstrated, the AmpG permease-defective background allows a better appreciation of the biological costs linked to the expression of different β-lactamases ( 25 ). In fact, the combination of AmpG disruption (as a paradigm of a peptidoglycan recycling-impaired strain) and AmpC hyperproduction (expressed from the multicopy plasmid pUCPAC) was also previously demonstrated to trigger significant P. aeruginosa virulence attenuation, in clear parallelism with PAΔDDh2Dh3 ( 21 ), and for these reasons, we also determined the LD 50 associated with pUCPAC versus pUCPAC* in PAΔAG.…”

“…Besides AmpC, the biological burden of expressing different acquired β-lactamases in P. aeruginosa has been shown to be highly variable, and whereas the production of the Ambler class B metallo-β-lactamase VIM-1 or the class A ESBL GES-1 did not have any impact on virulence, the production of various class D OXA-2-type enzymes entailed very interesting differences in the associated virulence attenuations in the Galleria mellonella invertebrate infection model, suggesting that modifications in the active center and, thus, in the hydrolytic behavior of OXA-2-like enzymes have a very important impact on fitness costs ( 25 ).…”

“…For this purpose, by site-directed mutagenesis, we assessed the role of AmpC β-lactamase activity per se in the virulence attenuation associated with its hyperproduction, providing clues to the pathways through which this enzyme is able to dampen bacterial fitness. Moreover, we wanted to ascertain whether several previously described AmpC variants involving P. aeruginosa resistance to CAZ-AVI and TOL-TAZ (T96I [PDC-222], G183D [PDC-322], and ΔG229-E247 [PDC-223]) ( 32 , 33 ) could carry differential biological burdens compared to the wild-type enzyme, similarly to what happens for OXA-2-like enzymes ( 25 ). Finally, we also applied this reasoning to the comparison of two closely related plasmid-encoded AmpC-type β-lactamases, namely, FOX-4 and FOX-8, which display different hydrolytic behaviors, with the former causing resistance to CAZ-AVI and TOL-TAZ and the latter showing a reduced capacity for CAZ hydrolysis compared to its progenitor FOX-3 ( 34 , 35 ).…”