Inactivation of the antimicrobial peptide LL-37 by pathogenic Leptospira
Introduction
Bacteria of the genus Leptospira are thin, elongated, and helical shaped Gram-negative spirochetes that may cause disease in humans and animals. Hitherto, 64 species of Leptospira classified into two major clades have been described. The P clade comprises pathogenic species, and the S clade is composed of free-living, saprophytic species [1]. More than 1 million cases of human leptospirosis are reported each year worldwide, with approximately 60 thousand deaths [2].
Once in the host, pathogenic leptospires spread very rapidly, and the great dissemination capacity of these spirochetes is attributed, among other factors, to their efficiency in moving through viscous media [3]. They also employ strategies to modulate host's microenvironment and innate immune responses, such as the ability to resist serum bactericidal activity [4,5].
The secretion of proteases with the potential to inactivate essential host proteins is an important tool used by several microorganisms during the colonization process. Pathogenic leptospires are no exception to this, and have been shown to secrete proteases capable of degrading a range of host molecules, including human complement system components as well as extracellular matrix molecules [[6], [7], [8]]. Certain pathogens of medical significance also display mechanisms of resistance to antimicrobial peptides, which help them to cause serious infections. Although rather resistant to proteolytic degradation [9], antimicrobial peptides can be efficiently cleaved and inactivated by bacterial proteases with a broad spectrum of activity [10]. These soluble, low molecular weight molecules play an important role in the innate immune response of humans, animals, and plants. By inhibiting the growth of microorganisms in the skin and mucous surfaces and their subsequent dissemination they contribute to natural immune responses in humans [11]. Upon insertion into membrane bilayers of their targets, antimicrobial peptides facilitate the elimination of microorganisms causing deleterious damage to them [12].
Cathelicidins are a family of antimicrobial peptides found in vertebrates. They are produced and stored as inactive pro-peptides in neutrophil granules [13,14], but are also produced by other immune cells including Natural Killer (NK) and mast cells, and by barrier epithelia [[15], [16], [17]]. Cathelicidins are constitutively expressed at low levels, but their expression is enhanced upon exposure to infectious agents leading to a local increase at the infection site. Approximately 30 members of the cathelicidin family have been described in mammals [18]. They have a conserved N-terminal cathelin domain and differ in their C-terminal regions, which are responsible for their known biological functions among which cytotoxic and antibacterial activities [19]. The only human cathelicidin identified so far, human cationic antimicrobial protein (hCAP18), has a molecular weight of 18 kDa, and consists of a signal peptide (30 aa residues), a highly conserved N-terminal domain (103 aa residues) and a C-terminal active domain (37 aa residues). To become active, the C-terminus with a predicted molecular mass of ~4 kDa corresponding to the peptide LL-37 is released from its precursor [20].
In the present study, we aimed to extend the range of investigation of possible targets for Leptospira proteases released to the extracellular milieu. We focused on antimicrobial peptides, notably on LL-37, which was shown to be particularly susceptible to degradation when in contact with culture supernatants of pathogenic Leptospira strains. Given that antimicrobial peptides are considered as potential therapeutic antibiotic candidates, increasing our current knowledge on the resistance mechanisms used by pathogenic Leptospira to counteract their effects may contribute to preventive or treatment approaches for leptospirosis.
Section snippets
Antimicrobial peptides
All antimicrobial peptides were chemically synthesized. LL-37 was purchased from Bachem (USA), and α-defensins HNP-1 and -2, and β-defensins 1 and 2 from Sigma-Aldrich™ (USA).
Leptospiral strains and growth conditions
The virulent strains Leptospira interrogans serovar Kennewick strain Fromm (LPF) and Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 (L1-130), the culture-attenuated Leptospira interrogans serovar Copenhageni strain 10A (Cop 10A) and Leptospira kirschneri serovar Cynopteri strain 3522C (Cynopteri), and the
Electrophoretic and proteolytic profiles of supernatant proteins secreted by Leptospira strains
In this work, 4 pathogenic and 2 saprophytic Leptospira strains were evaluated for their capacity to degrade human cathelicidin. The electrophoretic profiles of supernatant proteins of all 6 strains are shown in Fig. 1 A. Protein profiles of the two saprophytic strains are quite similar to each other but differ from those exhibited by the attenuated (Cop 10A and Cynopteri) or virulent (L1-130 and LPF) strains.
Proteolytic properties of the above-mentioned supernatants were evaluated by
Discussion
Antimicrobial peptides are part of the innate immune system of a variety of organisms including prokaryotes and eukaryotes [26]. Typically, they are short, amphiphilic, and positively charged molecules that induce cell damage mainly due to destabilization of the target cell membranes resulting from electrostatic interactions [27]. Certain pathogens of medical importance have mechanisms to counteract the action of antimicrobial peptides. Since leptospires are known to secrete proteases with a
Author statement
PNO and DSC: Performed the assays.
RMCC and LM: Purified the recombinant proteases.
GOS: Provided Leptospira strains.
MRF: Provided resources and contributed do data analysis.
EAWJ: Provided resources and contributed do data analysis.
MBH: Provided resources and contributed do data analysis.
ASB: Conceptualization and writing.
Declaration of competing interest
The authors declare they have no competing interest.
Acknowledgments
This research has benefited from grants provided by Fundação de Amapro à Pesquisa do Estado de São Paulo (FAPESP grant # 2018/12896-2). The authors also thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the fellowships granted to PNO (grant # 131434/2018-7), MBH (grant # 309145/2017-8) and ASB (grant # 305114/2017-4).
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