Extracellular Pneumococcal Serine Proteases Affect Nasopharyngeal Colonization.

Murtadha Q Ali,Sven Hammerschmidt,Gerhard Burchhardt,Nadin Henck,Robert Bolsmann,Franziska Voß,Thomas P Kohler,Andreas Wüst

doi:10.3389/fcimb.2020.613467

Murtadha Q Ali, Sven Hammerschmidt + Show 6 more

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https://doi.org/10.3389/fcimb.2020.613467

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Abstract

Streptococcus pneumoniae has evolved versatile strategies to colonize the nasopharynx of humans. Colonization is facilitated by direct interactions with host cell receptors or via binding to components of the extracellular matrix. In addition, pneumococci hijack host-derived extracellular proteases such as the serine protease plasmin(ogen) for ECM and mucus degradation as well as colonization. S. pneumoniae expresses strain-dependent up to four serine proteases. In this study, we assessed the role of secreted or cell-bound serine proteases HtrA, PrtA, SFP, and CbpG, in adherence assays and in a mouse colonization model. We hypothesized that the redundancy of serine proteases compensates for the deficiency of a single enzyme. Therefore, double and triple mutants were generated in serotype 19F strain EF3030 and serotype 4 strain TIGR4. Strain EF3030 produces only three serine proteases and lacks the SFP encoding gene. In adherence studies using Detroit-562 epithelial cells, we demonstrated that both TIGR4Δcps and 19F mutants without serine proteases or expressing only CbpG, HtrA, or PrtA have a reduced ability to adhere to Detroit-562 cells. Consistent with these results, we show that the mutants of strain 19F, which preferentially colonizes mice, abrogate nasopharyngeal colonization in CD-1 mice after intranasal infection. The bacterial load in the nasopharynx was monitored for 14 days. Importantly, mutants showed significantly lower bacterial numbers in the nasopharynx two days after infection. Similarly, we detected a significantly reduced pneumococcal colonization on days 3, 7, and 14 post-inoculations. To assess the impact of pneumococcal serine proteases on acute infection, we infected mice intranasally with bioluminescent and invasive TIGR4 or isogenic triple mutants expressing only CbpG, HtrA, PrtA, or SFP. We imaged the acute lung infection in real-time and determined the survival of the mice. The TIGR4lux mutant expressing only PrtA showed a significant attenuation and was less virulent in the acute pneumonia model. In conclusion, our results showed that pneumococcal serine proteases contributed significantly to pneumococcal colonization but played only a minor role in pneumonia and invasive diseases. Because colonization is a prerequisite for invasive diseases and transmission, these enzymes could be promising candidates for the development of antimicrobials to reduce pneumococcal transmission.

Highlights

Streptococcus pneumoniae is a Gram-positive, facultative human pathogen, and colonizes asymptomatically and highly successful mucosal epithelial surfaces of the upper respiratory tract (URT) (Kadioglu et al, 2008; Hilleringmann et al, 2015)
Binding to host cells is promoted by the interaction between bacterial proteins referred to as microbial surface components recognizing adhesive matrix molecules like enolase or the pneumococcal adherence and virulence factor A and B (PavA, PavB) and extracellular matrix (ECM) components such as fibronectin, vitronectin, thrombospondin-1, and plasminogen (Holmes et al, 2001; Rennemeier et al, 2007; Bergmann et al, 2009; Voss et al, 2012; Kanwal et al, 2017)
high-temperature requirement A (HtrA) consists of 393 amino acids that form a molecular weight of 42 kDa without a specific anchoring motif exhibiting two domains, the serine protease catalytic domain and the C-terminal PDZ domain

Summary

Introduction

Streptococcus pneumoniae (pneumococcus) is a Gram-positive, facultative human pathogen, and colonizes asymptomatically and highly successful mucosal epithelial surfaces of the upper respiratory tract (URT) (Kadioglu et al, 2008; Hilleringmann et al, 2015). Pneumococci use various strategies to interact with epithelial cell surface receptors Bacterial adhesins such as the pneumococcal surface protein C (PspC, known as CbpA), PavB, PsrP, or other adhesive pneumococcal surface components interact directly with host cell receptors (Pracht et al, 2005; Hammerschmidt, 2006; Orihuela et al, 2009; Kanwal et al, 2017; Weiser et al, 2018). Binding to host cells is promoted by the interaction between bacterial proteins referred to as microbial surface components recognizing adhesive matrix molecules like enolase or the pneumococcal adherence and virulence factor A and B (PavA, PavB) and extracellular matrix (ECM) components such as fibronectin, vitronectin, thrombospondin-1, and plasminogen (Holmes et al, 2001; Rennemeier et al, 2007; Bergmann et al, 2009; Voss et al, 2012; Kanwal et al, 2017). Pneumococci exploit hosts proteolytic activity such as plasmin to degrade mucosal and ECM components, thereby facilitating the tight interaction with host cells (Bergmann and Hammerschmidt, 2007; Bergmann et al, 2013)

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