Intracellular Staphylococcus Aureus Employs the Cysteine Protease

bioRxiv preprint doi: https://doi.org/10.1101/2020.02.10.936575; this version posted February 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 2 3 4 Intracellular Staphylococcus aureus employs the cysteine protease 5 staphopain A to induce host cell death in epithelial cells 6 7 Kathrin Stelzner1, Tobias Hertlein2, Aneta Sroka3, Adriana Moldovan1, Kerstin 8 Paprotka1, David Kessie1, Helene Mehling1, Jan Potempa3,4, Knut Ohlsen2, Martin J. 9 Fraunholz1, Thomas Rudel1* 10 11 1 Chair of Microbiology, University of Würzburg, Würzburg 97074, Germany 12 2 Institute for Molecular Infection Biology (IMIB), University of Würzburg, Würzburg 13 97080, Germany 14 3 Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 15 Kraków, Poland 16 4 Department of Oral Immunology and Infectious Diseases, University of Louisville 17 School of Dentistry, Louisville, KY, USA 18 19 * Corresponding author 20 E-mail: [email protected] 21 22 23 24 25 26 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.10.936575; this version posted February 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 27 Abstract 28 Staphylococcus aureus is a major human pathogen, which can invade and survive in 29 non-professional and professional phagocytes. Intracellularity is thought to contribute 30 to pathogenicity and persistence of the bacterium. Upon internalization by epithelial 31 cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the 32 cytosol and induce host cell death. Here, we identified a staphylococcal cysteine 33 protease to induce cell death by intracellular S. aureus after translocation into the 34 host cell cytoplasm. We demonstrated that loss of staphopain A function leads to 35 delayed onset of host cell death and prolonged intracellular replication of S. aureus in 36 epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated 37 intracellular killing of the host cell even in the absence of detectable intracellular 38 replication. Moreover, staphopain A contributed to efficient colonization of the lung in 39 a mouse pneumonia model. Our study suggests that staphopain A is utilized by S. 40 aureus to mediate escape from the host cell and thus contributes to tissue 41 destruction and dissemination of infection. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.10.936575; this version posted February 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 42 Author Summary 43 Staphylococcus aureus is a well-known antibiotic-resistant pathogen that emerges in 44 hospital and community settings and can cause a variety of diseases ranging from 45 skin abscesses to lung inflammation and blood poisoning. The bacterium 46 asymptomatically colonizes the upper respiratory tract and skin of about one third of 47 the human population and takes advantage of opportune conditions, like 48 immunodeficiency or breached barriers, to cause infection. Although S. aureus is not 49 regarded as a professional intracellular bacterium, it can be internalized by human 50 cells and subsequently exit the host cells by induction of cell death, which is 51 considered to cause tissue destruction and spread of infection. The bacterial 52 virulence factors and underlying molecular mechanisms involved in the intracellular 53 lifestyle of S. aureus remain largely unknown. We identified a bacterial cysteine 54 protease to contribute to host cell death mediated by intracellular S. aureus. 55 Staphopain A induced killing of the host cell after translocation of the pathogen into 56 the cell cytosol, while bacterial proliferation was not required. Further, the protease 57 enhanced survival of the pathogen during lung infection. These findings reveal a 58 novel, intracellular role for the bacterial protease staphopain A. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.10.936575; this version posted February 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 59 Introduction 60 Staphylococcus aureus is a Gram-positive bacterium frequently colonizing human 61 skin and soft tissue, primarily the anterior nares, as part of the normal microflora [1]. 62 However, in hospital- as well as in community-settings it arises as an opportunistic 63 pathogen causing a plethora of diseases ranging from local, superficial skin 64 infections, wound infections and abscesses to invasive, systemic diseases like 65 osteomyelitis, pneumonia, endocarditis or sepsis [2]. This can be largely attributed to 66 its vast array of virulence factors [3]. In addition, the emergence and rapid spread of 67 methicillin-resistant S. aureus (MRSA) strains makes this pathogen particularly 68 difficult to treat and leads to significant morbidity and mortality worldwide [4]. 69 Whereas S. aureus is originally considered an extracellular pathogen, substantial 70 evidence exists that it is able to invade non-phagocytic mammalian cells, like 71 epithelial and endothelial cells, osteoblasts, fibroblasts or keratinocytes [e.g. 5, 6-8], 72 as well as to survive internalization by professional phagocytes [e.g. 9, 10, 11]. 73 Several studies demonstrate the existence of intracellular S. aureus in tissue and 74 phagocytic cells in vivo [e.g. 12, 13-16]. Invasion of tissue cells is facilitated by 75 numerous different bacterial adhesins and followed by escape from the bacteria- 76 containing vacuole and cytosolic replication [reviewed in 17, 18, 19]. In professional 77 phagocytes, intracellular S. aureus is able to resist the antimicrobial attack by the 78 host cell and replication occurs within phagosomes. In both cell types, the pathogen 79 eventually kills the host cell from within and a new infection cycle can be initiated. 80 Numerous rounds of internalization and release may lead to excessive cell and tissue 81 destruction, persistence and dissemination of infection, immune evasion and 82 protection from antibiotic treatment [20-22]. 83 Intracellular pathogens may induce killing as a strategy to exit from the host cell. 84 Induction of programmed cell death, such as apoptosis, necroptosis or pyroptosis, or 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.10.936575; this version posted February 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 85 the damaging of host cell-derived membranes such as endosomal, vacuolar and 86 plasma membrane have been demonstrated as mechanisms [reviewed in 23]. 87 Reports on the molecular mechanisms underlying S. aureus induced host cell killing 88 are rather inconsistent, which likely arises from the diversity of virulence factors 89 within S. aureus. Some studies describe induction of apoptosis in professional as well 90 as non-professional phagocytes [e.g. 24, 25-27], while others observed hallmarks of 91 both apoptosis and necrosis [11, 28]. In epithelial cells, an autophagy-associated cell 92 death was discovered [29], whereas in primary human polymorphonuclear 93 neutrophils (PMNs) intracellular S. aureus induced a necroptotic cell death [30, 31]. 94 The existence of different types of host cells and a multitude of different S. aureus 95 strains further impedes the elucidation of the process and bacterial virulence factors 96 involved therein. 97 To date, several S. aureus virulence factors have been linked to intracellular 98 cytotoxicity. In non-professional phagocytes the hemolytic α-toxin was identified as a 99 key factor mediating intracellular cytotoxicity [32, 33], whereas the bi-component 100 leukotoxin LukAB (also known as LukGH) was shown to induce cell lysis after uptake 101 of S. aureus by professional phagocytes [34-37]. Some reports also connected 102 Panton-Valentine-leukotoxin (PVL) [35, 38, 39] or phenol-soluble modulins (PSMs) 103 [40, 41] with host cell killing by intracellular S. aureus. 104 Beside these toxins, S. aureus secretes several proteases which were shown to 105 contribute to virulence of the pathogen [42] and whose role in the intracellular lifestyle 106 of the pathogen has not been investigated so far. S. aureus possesses two papain- 107 like cysteine proteases, staphopain A (ScpA) and staphopain B (SspB), which have 108 almost identical three-dimensional structures, despite sharing limited primary 109 sequence identity [43, 44]. Both proteases are highly conserved among S. aureus 110 isolates [45] and are expressed in the respective operons, scpAB and sspABC, 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.10.936575; this version posted February 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 111 together with their endogenous inhibitors, the staphostatins ScpB and SspC, which 112 protect the bacteria from proteolytic degradation [46-48]. Staphopain A is secreted as 113 zymogen and activated by autolytic cleavage once outside the bacterial cell [49]. By 114 contrast, activation of staphopain B is the result of a proteolytic cascade initiated by 115 aureolysin-mediated cleavage and activation of the V8 protease (SspA) which in turn 116 processes SspB [47, 50]. In vitro experiments revealed a very broad activity of both 117 enzymes on the destruction of connective tissue, the evasion of host immunity and 118 the modulation of biofilm integrity [51-57]. A staphopain A-like protease with similar 119 functions, called EcpA, is also expressed by S. epidermidis and other coagulase- 120 negative staphylococci [42, 58, 59]. In contrast, orthologues of the sspABC operon 121 have only been identified in S. warneri [60, 61]. 122 In this study, we identified a novel role of S.

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