Molecular Analysis of the Contribution of Alkaline Protease A and Elastase B to the Virulence of Pseudomonas aeruginosa Bloodstream Infections.

Margalida Mateu-Borrás,Antonio Doménech-Sánchez,Antonio Oliver,Sebastián Albertí,Laura Zamorano,Alex González-Alsina,Irina Sánchez-Diener

doi:10.3389/fcimb.2021.816356

Abstract

Pseudomonas aeruginosa is a major cause of nosocomial bloodstream infections. This microorganism secretes two major proteases, alkaline protease A (AprA) and elastase B (LasB). Despite several in vitro studies having demonstrated that both purified proteases cleave a number of components of the immune system, their contribution to P. aeruginosa bloodstream infections in vivo remains poorly investigated. In this study, we used a set of isogenic mutants deficient in AprA, LasB or both to demonstrate that these exoproteases are sufficient to cleave the complement component C3, either soluble or deposited on the bacteria. Nonetheless, exoprotease-deficient mutants were as virulent as the wild-type strain in a murine model of systemic infection, in Caenorhabditis elegans and in Galleria mellonella. Consistently, the effect of the exoproteases on the opsonization of P. aeruginosa by C3 became evident four hours after the initial interaction of the complement with the microorganism and was not crucial to survival in blood. These results indicate that exoproteases AprA and LasB, although conferring the capacity to cleave C3, are not essential for the virulence of P. aeruginosa bloodstream infections.

Highlights

Pseudomonas aeruginosa is one of the most common Gram-negative organisms causing nosocomial bacteremia
Previous in vitro studies have shown that purified alkaline protease AprA and LasB elastase degrade C3 (Hong and Ghebrehiwet, 1992; Laarman et al, 2012)
Incubation of the cell-free supernatants of cultures from PA14 or the mutants PA14DaprA and PA14DlasB with C3 generated a ~100-kDa a-chain (a”-chain) resulting from cleavage of the 111kDa a chain (Figure 1A), while the b chain remained intact (Supplementary Figure S1). This a”-chain was absent from the samples incubated with the cell-free supernatant of the culture from the double mutant or the control incubated with LB alone, showing that the presence of this chain was due to the P. aeruginosa-mediated cleavage of C3 rather than to the physiological activation of C3, which would result in the formation of a smaller a-chain (a’)

Summary

Introduction

Pseudomonas aeruginosa is one of the most common Gram-negative organisms causing nosocomial bacteremia. The reasons for the mortality of P. aeruginosa bacteremia are multifactorial and include the intrinsic virulence of the microorganism and some underlying host conditions It is remarkable that a large number of deaths occur within the first 24–72 hours after P. aeruginosa infection (Hattemer et al, 2013; Peña et al, 2013), suggesting an inefficient function of the early innate immune mechanisms of the host. C3 is the most abundant complement protein within serum It is a 190 kDa protein, composed of two glycoprotein chains (a and b) associated through disulphide bonds. It is required for each of the three complement-activating pathways: classical, lectin, and alternative. C3b is involved in the formation of C5 convertases that cleave C5 and trigger the subsequent deposition of the lytic pathway complement components to form the membrane attack complex, which may cause the killing of this microorganism

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Results

Conclusion