Development of a New N-Terminomic Method to Study the Pathodegradome of theStaphylococcus aureusV8 Protease in Human Neutrophils

30 Staphylococcus aureus is a notorious human pathogen that relies on an array of 31 virulence factors to engender infection and evade the host-immune system. Among these are 32 the secreted proteases, which promote pathogenesis by degrading host proteins and 33 modulating host-defenses. Human neutrophils play a pivotal role in these defenses, acting as 34 the first responders against invading bacteria. While many S. aureus effectors of virulence have 35 been shown to target leukocytes, there is limited knowledge on how the extracellular proteases 36 modulate neutrophil fate. Typically, protease substrates have been identified in isolated 37 settings using one at a time approaches; with neutrophil targets few and far between. Herein, 38 we have developed a novel N-terminomic methodology termed TAGS-CR that can facilitate 39 global substrate characterization in streamlined manner. We thus present the application of 40 TAGS-CR to unravelling the human neutrophil pathodegradome of the S. aureus V8 protease. In 41 so doing, we captured ~350 V8 targets, revealing critical insight into how this virulence factor 42 can modulate neutrophil functionality on various levels relevant to S. aureus disease 43 progression. We recorded cleavage of proteins necessary for neutrophil adhesion and 44 migration, a fundamental process necessary for pathogen clearance. Furthermore, we highlight 45 V8 cleavage of proteins involved in important neutrophil defense tactics, such as degranulation 46 and reactive oxygen species production. This protease may also facilitate bacterial 47 dissemination via the intentional activation of neutrophil apoptosis. Collectively, this work 48 deepens our understanding of host-pathogen interaction and begins to unravel how S. aureus 49 proteases can induce immune dysregulation through the targeting of leukocytes. 50 51 Importance 52 During infection Staphylococcus aureus must engage and evade the host immune 53 system in order to successfully cause disease. As neutrophils represent the frontline of defense 54 against invading S. aureus cells, it becomes increasingly important to decode how this 55 bacterium subverts their host-defense tactics. While the contributing role to neutrophil 56 engagement for many S. aureus virulence factors have been elucidated, the effects of their 57 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint proteases remain largely unclear. Here, we present a novel method for global protease 58 substrate identification, TAGS-CR, and use it to identify S. aureus V8 protease targets in human 59 neutrophils. These include factors that not only govern general neutrophil function but 60 moreover, their defense mechanisms, such as migration, degranulation, oxidative defense, 61 phagocytosis and apoptosis. 62 63 64

Staphylococcus aureus, V8 protease, Neutrophils, Degradomics, TAGS-CR, Immune 65 dysregulation. 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint

85 Staphylococcus aureus is an adept human pathogen that is uniquely skilled at causing a 86 wide array of diseases, ranging from skin and soft tissue infections, to more invasive conditions, 87 such as osteomyelitis, endocarditis, pneumonia, and bacteremia (1). The ability to engender 88 these various infections stems from a cadre of tightly regulated virulence factors produced by S. 89 aureus, ranging from adhesins to hemolysins, toxins, lipases , proteases and other exoenzyme s 90 (2). These virulence determinants function to not only wreak havoc on host tissues but are also 91 geared toward immune evasion and hijacking of the innate immune system (3). 92 In keeping with this, S. aureus is known to target polymorphonuclear leukocytes (PMNs 93 or neutrophils), which serve as professional innate immune phagocytes in the host (4). PMNs are 94 considered the primary line of defense against S. aureus infections in humans and are thus 95 indispensable if pathogen clearance from the host is to be successful ( 5). Once at the infection 96 site neutrophils can engulf opsonized S. aureus and employ mechanisms geared towards 97 pathogen killing. Perhaps the most effective antibacterial tactic is the production of reactive 98 oxygen species (ROS) via the NADPH oxidase complex (6,7). These molecules function to not only 99 elicit damage directly to the bacterial membrane and intracellular components, but also enhance 100 the immune response of PMNs (8). In addition to ROS, neutrophils also promote pathogen 101 clearance via degranulation (9). 102 Consequently, neutrophil extravasation and defense present opportunities that S. aureus 103 is well known to take advantage of (3). Indeed, it possesses a repertoire of virulence factors that 104 have been shown to inhibit neutrophil activation and chemotaxis, including chemotaxis inhibitory 105 protein (CHIPS), formyl -peptide receptor Like -1 inhibitory protein (FLIPr) , and staphylococcal 106 superantigen proteins SSL3, SSL4, SSL5 and SSL10 (10-12). In addition, it also possesses immune 107 evasion proteins such as staphylococcal enterotoxin-like toxin X (SElX) , extracellular adherence 108 protein (Eap), and again SSL5 as well as SSL11, which are all geared towards hindering neutrophil 109 adhesion to host epithelial surfaces (12). If neutrophils reach the site of infection, their function 110 can be further inhibited by S. aureus via strategies to prevent opsonophagocytosis via 111 complement inhibitory proteins, as well as blocking IgG antibodies bound to its surface (12). 112 Further, if engulfed by PMNs, S. aureus can produce another set of immune evasion molecules 113 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint to circumvent the antimicrobial activities of the phagolysosome . These include superoxide 114 dismutases (SodM and SodA), catalase (KatA), staphylococcal peroxidase inhibitor (SPIN), 115 Flavohemoprotein (Hmp), alkyl hydroperoxide reductase (AhpC), and staphyloxanthin, to 116 neutralize ROS (13-18). Furthermore, staphylokinase (SAK), O-acetyltransferase A (OatA) and Eap 117 will inhibit bacterial destruction by antimicrobial peptides (19-21). 118 Despite knowledge of how these virulence factors target immune cells, the role of the 119 secreted proteases, and how they modulate neutrophil function, is far less well understood (22). 120 All strains of S. aureus produce 4 major extracellular proteases: a metalloproteinase (Aureolysin, 121 aur), a glutamyl endopeptidase ( sspA or the V8 serine protease) , and two cysteine proteases 122 known as Staphopain A ( scpA) and Staphopain B ( sspB) (23). Additionally, depending on the 123 sequence type, strains also produce up to 9 serine-protease-like enzymes known as the Spls (24). 124 Contribution of these exoenzymes to infection is dual sided, with S. aureus proteases regulating 125 the progression of infection by modulating the stability of other virulence factors, whilst at the 126 same time having their own role in cleavage of host defense molecules (25,26). In a few instances, 127 staphylococcal proteases have been shown to inhibit neutrophil function, for example 128 staphopain A cleaves chemokine receptor 2 (CXCR2) resulting in diminished recruitment ( 27). 129 Staphopain B also induces cleavage of CD11b and CD31, impairing neutrophil activation and 130 survival, respectively (28,29). While V8, Staphopain B and Aureolysin were found to be produced 131 following neutrophil phagocytosis, knowledge of their phagolysosomal targets is at best limited 132 (30); with only the neutrophil antibacterial peptide LL-37 revealed to be a substrate (31,32). 133 Previously, our group deployed N -Terminomic methodologies as a global strategy to 134 identify targets of the V8 protease in human serum (33). Approximately 90 substrates were 135 identified in this study , with many found to be directly relevant to S. aureus pathogenesis, 136 including proteins of the clotting cascade, complement system and protease inhibition networks 137 (33). Herein, we use a newly developed N -terminomic methodology to explore the 138 pathodegradome of the V8 protease in human neutrophils. In so doing, we were able to enhance 139 target identification, capturing 346 V8 substrates. Our results demonstrated that the V8 protease 140 adopts a holistic approach to neutrophil engagement and targets multiple aspects of neutrophil 141 functionality, ranging from migration to phagocytosis, intracellular defenses and apoptosis. 142 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint

and DISCUSSION 143 Development of the TAGS-CR N-terminomic methodology: We previously published the use of 144 TAILS N-terminomics to study the pathodegradome of the V8 protease from Staphylococcus 145 aureus (33). Herein, we have developed terminal amine guanidination of substrates-charge 146 reversal (TAGS-CR) as a novel N -terminomic methodology that uses an alternative enrichment 147 strategy avoiding the use of single-use polymers. Our workflow is outlined in Figure 1 and uses a 148 combination of previously defined chemistries for the identification of protease substrates 149 (34,35). Guanidination (Gd) of lysine ε-amines and N-terminal α-amines was our chosen labelling 150 strategy as this method not only negates digestion inefficiency seen with more traditional 151 approaches, such as d imethylation or acetylation modifications as a result of blocked lysine 152 residues, but also increases ionization efficiency of peptides ( 34). Gd-labeled samples under go 153 reduction, alkylation, and tryptic digest prior to downstream enrichment via charge reversal (CR) 154 of internal peptides and strong cation exchange (SCX) (35,36). Post digest, peptides undergo a 155 series of incubation steps whereby internal, unlabeled tryptic peptides gain a negative charge via 156 sulfation. Strong cation exchange then facilitates enrichment of Gd-labelled peptides, which had 157 amines already converted to guan idinyl groups prior to tryptic digest. Thus, the native - or neo- 158 (i.e. caused by proteolysis prior to trypsini zation) N-termini are retained during SCX, but 159 negatively charged tryptic peptides can no longer bind to the SCX matrix , thus facilitating their 160 removal. 161 162 Uncovering V8 substrates within neutrophil proteomes: Utilizing TAGS-CR, we sought to expand 163 our studies with the V8 protease of S. aureus , deploying our new method to explore the 164 pathodegradome of human neutrophils. Accordingly, the proteomes of differentiated HL-60 cells 165 were treated with V8 protease and, employing a binary comparison of N-termini between treated 166 and untreated samples, TAGS -CR identified 346 V8 protein substrates (Table S1). Of these, we 167 captured 454 cleavage events unique to the V8 treated condition (Table S2). It is well established 168 that the V8 protease cleaves primarily at the carboxyl side of glutamic acid (E) and to a lesser 169 extent aspartic acid (D) residues (37). As such, we sought to initially validate our V8 targets via 170 analysis of the amino acids surrounding the identified cleavage sites (Figure 2A and 2B). Of the 171 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint 454 neo-N-termini, glutamic acid (E) accounted for ~90% of the residues upstream of the scissile 172 bond at position P1 ( Figure 2A). Sequence analysis with ICELOGO (38) was also used to assess 173 sequence conservation surrounding the cleavage position (P5 -P5’) (Figure 2B). In keeping with 174 the specificity of the V8 protease, we found that only glutamic acid was significantly increased at 175 P1 (Figure 2B). We also observed a significant increase in alanine and glycine residues at P1’, 176 leucine residues at position P2 and valine residues at P4 ’ (Figure 2B). Our previous study of V8 177 targets in human serum revealed a similar trend in amino acids at these positions, indicating a 178 likely consensus sequence for V8 protease substrate preference (33). Additionally, in our past 179 (33) and present study we noted an apparent inhibitory effect of proline on the activity of the V8 180 enzyme due to decreased frequency at position s P3 and P1’. The inhibitory effect of proline on 181 not only V8 activity but protease activity in general has previously been recorded in the MEROPS 182 database, and in a number of in vitro experiments (39-41). The similarity in cleavage consensus 183 sequence between our study and those previous, indicates that changing methods from TAILS to 184 TAGS-CR did not bias identification of cleavage sites, offering additional validation of TAGS -CR, 185 while providing a new and diverse set of host substrates for V8. 186 187 Ingenuity pathway analysis highlights host canonical signaling pathways targeted by V8: Using 188 the Ingenuity Pathway Analysis (IPA) software, we were able to highlight canonical host signaling 189 pathways to which V8 leukocyte targets belonged . Here, we identified 920 signaling cascades 190 potentially being modulated by the S. aureus V8 protease (Table S3). Among these included , 191 leukocyte extravasation signaling, integrin cell surface interaction s, neutrophil degranulation, 192 Rac2 signaling and other oxygen -dependent ROS production pathways, phagocytosis and 193 phagosomal maturation, and finally, the intrinsic pathway for apoptosis (Table S3 and Figure 3). 194 This analysis suggests that the V8 protease can hinder neutrophil functionality on many different 195 levels relevant to S. aureus pathogenesis. 196 197 TAGS-CR highlights V8 targets essential for leukocyte extravasation: Neutrophil recruitment is 198 an integral process typically involving various receptors on the surface of cells ( 42). Once 199 neutrophils become activated, they adhere and migrate through host tissues to different sites of 200 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint infection via transcellular or paracellular routes , in a process termed neutrophil extravasation 201 (42). TAGS -CR captured V8 induced cleavage of various receptor proteins belonging to this 202 signaling pathway , such as intracellular adhesion molecule 3 (ICAM3), guanine nucleotide -203 binding protein G(i) subunit alpha-2 (GNAI2), transforming protein RhoA (RHOA), Ras-related C3 204 botulinum toxin substrate 2 (RAC2), moesin (MSN), and integrin alpha -L (ITGAL). Upon closer 205 assessment, the primary influence of V8 on this process is seemingly geared towards modulating 206 the adhesion dynamics of neutrophils. For example, moesin belongs to a group of adaptor 207 proteins, collectively termed ERM (ezrin/radixin/moesin), that influence leukocyte response (43). 208 Previous studies have demonstrated moesin and moesin/ezrin knockout neutrophils present 209 with abrogated adhesive abilities ( 44). V8 could potentially mimic this effect as cleavage, 210 occurring at position 396 in the alpha helix domain could diminish functional activity of moesin. 211 In its active and subsequently unfolded state, this protein acts as a bridge between actin 212 filaments and the plasma membrane via their interaction with opposing ends of this molecule 213 (43). While this protein’s FERM domain mediates attachment to the plasma membrane, cleavage 214 at any position would destroy the linkage effect of moesin, resulting in dissociation of actin and 215 plasma membrane structures with the overall effect of potentially hindering neutrophil adhesion. 216 Similarly, we also captured Rac2 and RhoA as V8 substrates. These proteins belong to the Rho 217 family of small GTPases, and , while they are multifunctional, regulating numerous neutrophil 218 activities, importantly they also control neutrophil adhesion ( 45,46). Moreover, this process of 219 neutrophil arrest could further be modulated by V8 cleavage of ICAM3 and ITGAL, both of which 220 facilitate neutrophil adherence to epithelial surfaces via binding to their respective ligands (47). 221 Integrins, like ITGAL, have been found to play pivotal roles in promoting not only 222 neutrophil adhesion and movement but also effector functions during inflammatory conditions 223 such as S. aureus infection (48). V8 cleaves ITGAL (CD11a) at positions 477 and 510, located in 224 the FG-GAP-5 and FG -GAP-6 repeats, respectively. The N -terminal region of the integrin alpha 225 subunit contains seven FG -GAP repeats, of about 60aa each, that fold to form a beta propellor 226 domain important for ligand binding (49). While both magnesium and calcium ions bind this beta 227 propellor domain and activate the integrin alpha subunit, V8 activity would disrupt calcium 228 binding as cleavage of this molecule occurs in the middle of a calcium binding motif present in 229 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint the FG-GAP-6 repeat (Protein Data Bank in Europe Knowledge Base, PDBe-KB) (49). While TAGS-230 CR only captured cleavage of the integrin alpha subunit, CD11a, these molecules are typically 231 heterodimeric in form (50). As such, the ITGAL chain (CD11a) will combine with the integrin beta 232 2 chain (ITGB2 or CD18) to form the leukocyte function-associated antigen (LFA)-1, also known 233 as a β2 integrin, a complex integral to neutrophil migration upon ligand binding ( 50). Highly 234 expressed on the surface of neutrophils once activated by cytokines, LFA -1 will bind to ICAM -1 235 found on the surface of endothelial cells, facilitating adhesion prior to migration (50). V8 236 mediated cleavage of the ITGAL chain could work to dismantle this complex, perpetuating S. 237 aureus survival by hindering bacterial clearance due to an insufficient number of neutrophils at 238 the site of infection. The (LFA)-1 complex has long been a molecule of interest as defects in its 239 expression cause leukocyte adhesion deficiency (LAD) -1 syndrome in the host ( 50). In patients 240 suffering with LAD -1, impairment of the adhesive ability of leukocytes has been linked to 241 mutations in the CD18 subunit of the complex resulting in nonfunctional LFA -1 ( 50,51). 242 Importantly, patients suffering with LAD -1 syndrome experience recurrent bacterial infections, 243 along with detrimental health effects such as painful lesions that fail to heal ( 50,51). Recurrent 244 bacterial infections are a hallmark of S. aureus disease and are often seen in patients with 245 leukocyte defects (52). S. aureus proteases may therefore contribute to the occurrence of these 246 continuing infections via cleavage of the ITGAL chain by V8. This may compromise the activity of 247 the LFA-1 complex, leading to a dampened neutrophil response during infection. 248 An important consideration with proteomic studies such as ours is that rigorous validation 249 of targets must be undertaken. As such , we confirmed V8 cleavage of ITGAL via immunoblot 250 (Figure 4A). In untreated neutrophil proteomes, we observed a ~129kDa band corresponding to 251 the predicted molecular weight of this protein , as well as a n additional band accounting for a 252 glycosylated form . Proteolytic cleavage of ITGAL by V8 at residue 510 would give rise to a C -253 terminal fragment ~72kDa in size – which was observed in the immunoblot. (Figure 4A). 254 255 V8 further hampers neutrophil migration by targeting integrin activators: Leukocyte defects 256 come in many forms and range from type I, described above, to type III (53). While mutations in 257 the ITGB2 gene cause LAD type I, type III is caused by mutations in the FERMT3 gene (54,55). 258 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint Patients suffering with LAD III similarly experience recurrent bacterial infections along with other 259 symptoms including skin and soft tissue lesions and uncontrollable bleeding ( 54,55). FERMT3 260 encodes the fermitin family homolog 3 (K indlin-3) protein product, which was found to be 261 cleaved by the V8 protease (Table S1 and S2). In tandem with talin-1, notably also a V8 substrate, 262 kindlin-3 is responsible for ‘inside out’ signaling to activate the LFA -1 integrin complex (56). This 263

in a conformational change, ultimately allowing this molecule to interact with its binding 264 partners and arrest to the vascular endothelium prior to slow rolling along the surface and 265 subsequent transmigration ( 53,56). Previous studies have described in vivo data where 266 neutrophil deficiency in kindlin-3 resulted in attenuation of neutrophil arrest ( 56). In our study, 267 we hypothesize that V8 cleavage of this molecule could have similar effects. Two V8 cleavage 268 events were detected in Kindlin-3, with start position s 336 and 500, both corresponding to the 269 FERM domain. Similar to ITGAL, kindlin-3 combines with a beta integrin subunit, in this case 270 ITGB1, ITGB2 or ITGB3 (54). As such, the FERM domain of kindlin-3 induces activation of the beta 271 subunit and complex formation ( 54); thus, V8 cleavage would disrupt this interaction. 272 Interestingly, in patients with the LAD -III deficiency, a FERMT3 gene mutation was seen 273 corresponding to position 552 in the protein product, also found in the FERM domain (54). It was 274 hypothesized that this mutation would hinder the binding capabilities of the FERM domain to its 275 beta integrin interaction partner ( 54). While mutations are not akin to cleavage, our data 276 highlights multiple avenues by which neutrophil arrest and subsequent migration may be 277 targeted by the S. aureus V8 protease. 278 During the validation of cleavage for this pathway , immunoblotting with antibodies 279 against kindlin-3 revealed a band corresponding to the full -length protein at ~75kDa in both V8 280 treated and untreated proteomes, however with a higher intensity in the latter, as expected since 281 no enzyme was present (Figure 4B ). Studies have shown this protein to be heavily 282 phosphorylated at approximately 30 sites ( 57), thus possibly accounting for the additional band 283 directly below the full-length protein (Figure 4B). Our immunoblot also presented an unexpected 284 banding pattern between ~3 0-50kDa in both V8 treated and untreated conditions, possibly 285 indicating that native proteolysis of kindlin -3 is occurring in the neutrophil proteome, 286 independent of V8 treatment (Figure 4B). In line with this, kindlin-3 has been shown to be cleaved 287 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint by calpain, an intracellular cyst eine protease found abundantly in the HL60 promyelocytic 288 leukemia cell line from which our neutrophil cells were derived ( 57-60). Calpain cleavage of 289 kindlin-3, found at position 373, would yield two fragments, one of which would be ~4 2kDa - 290 directly corresponding to the fragment observed ( Figure 4B). However, independent to calpain 291 activity, we were still able to validate V8 cleavage of kindlin-3 at positions 336 and 500. In treated 292 samples, we observed a cluster of 3 unique bands, one of which directly aligns with the V8 293 liberated N-terminal fragment at ~ 18kDa outlined in the protein schematic ( Figure 4B). Likely, 294 the surrounding bands at ~15kDa and ~2 3kDa may correspond to modified versions of the 295 specific V8 liberated fragment. 296 297 Dysregulation of neutrophil degranulation by V8: Despite the many efforts of pathogens like S. 298 aureus to prevent neutrophil migration, these workhorses of immunity can still reach the site of 299 infection to engulf and clear invading bacteria from the host (61). To facilitate intracellular killing, 300 neutrophils undergo degranulation and will generate and release potent microbicidal molecules 301 including superoxides, other reactive oxygen species (ROS), as well as degradative enzymes and 302 peptides (22,52,67). IPA highlighted 41 proteins involved in the process of neutrophil 303 degranulation, as targets of V8 (Table S3 ). U pon further analysis we found several of these 304 proteins to be directly implicated in pathogen mediated killing , either serving as an effector 305 molecule or their precursor. Among them were cathepsin C, a degradative lysosomal cysteine 306 peptidase known to promote pathogen clearance whilst also serving as an activator of other 307 important neutrophil serine proteases , including elastase, proteinase 3 and cathepsin G, all 308 constituting as azurophilic granules that exhibit a similar effect (62). Thus, our data suggests that 309 cleavage of this molecule by V8 , and therefore a potential dampening of its activity, may 310 compromise the host ’s ability to defend against S. aureus. This notion is further supported by 311 previous research revealing that neutrophils lacking olfactomedin 4 , a negative regulator of 312 cathepsin C activity, demonstrated increased intracellular killing of S. aureus (62). Moreover, in 313 vivo, mice carrying these mutated immune cells experienced enhanced clearance of S. aureus, 314 largely owing to an increase in activity of this lysosomal exopeptidase , highlighting its essential 315 role in innate immune defense (62). 316 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint Beyond cathepsin C, TAGS -CR also captured V8 cleavage of the endosomal adaptor 317 protein p14 (LAMTOR2). I mmunodeficiency in this protein has been shown to culminate in 318 congenital neutropenia, altered microarchitecture of azurophilic granules, and importantly 319 decreased ability for pathogen clearance within the phagosome (63). Whilst V8’s negative impact 320 on the azurophilic granules of human leukocytes may be indirect through the targeting of 321 cathepsin C and the endosomal adaptor protein p14, we also observed direct degradation of 322 these key molecules via cleavage of myeloperoxidase (MPO). Myeloperoxidase is a heme driven 323 antimicrobial enzyme that facilitates catalysis of hydrogen peroxide to hypochlorous acid, a 324 reactive oxygen species (ROS) (64,65). Participating in oxidative defense, this potent ROS causes 325 irreparable damage to invading S. aureus (66). We observe V8 cleaving MPO at position 412, 326 downstream of its heme binding cluster at residues 495, 498-502 and 505 (PDBe-KB). Given the 327 ferric form of MPO is required to drive the catalytic cycle of this enzyme (65), V8 cleavage near 328 these residues would act to destabilize this molecule and decrease HOCl production. To 329 emphasize how important MPO inhibition is for S. aureus pathogenesis, this bacterium encodes 330 an additional secreted virulence factor , staphylococcal peroxidase inhibitor or SPIN, that is a 331 specific inhibitor of MPO activity (66). As such , in addition to SPIN, our findings suggest an 332 alternative avenue of protection for S. aureus against MPO -dependent killing elicited by V8. 333 Collectively, our data suggests that V8 may assist in mitigating bactericidal activities of the 334 neutrophil phagosome, potentially allowing S. aureus to survive intracellularly and further 335 disseminate to different niches within the host. 336 In support of this, we present immunoblot validation of myeloperoxidase cleavage (Figure 337 4). MPO is a homodimer, with each identical monomer having a heavy chain (60kDa) and a light 338 chain (15kDa) (68,69). In the untreated control, immunoblotting revealed the heavy chain of 339 myeloperoxidase at ~60kDa (Figure 4C). In V8 treated proteomes, we still observed the presence 340 of the heavy chain, however, an additional, major cleavage product appeared at ~38kDa (Figure 341 4C). This corresponds to the C -terminal fragment of myeloperoxidase produced following V8 342 cleavage of the heavy chain at position 412 (Figure 4C). 343 344 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint Oxygen-dependent host-defense mechanisms are vulnerable to V8 activity: Continued 345 targeting of ROS production by V8 was observed via cleavage of multiple proteins involved in 346 activation of the NADPH or phagocyte oxidase complex that governs respiratory burst and 347 superoxide production within neutrophil phagolysosomes (6). Neo -N-termini arising from V8 348 treatment were found belonging to not only downstream enzymes modulating the activity of the 349 complex, such as pyruvate kinase M2 (PKM2), protein kinase C alpha type (PKC -alpha), and Ras-350 related C3 botulinum toxin substrate 2 (Rac2), but also a physical subunit of the NADPH oxidase 351 complex, putative neutrophil cytosol factor 1C (p47 phox or NCF -1C) ( 70-73). NADPH oxidase 352 activity is ultimately dependent on glycolysis and glycolytic intermediates to trigger the assembly 353 of its subunits and subsequent ROS production ( 70). Herein, we see a potential inhibitory effect 354 of the V8 protease on this process as PKM2 is the rate limiting enzyme , and once allosterically 355 activated, controls ROS production via mediation of glycolytic intermediates ( 70). Emphasizing 356 the importance of this molecule to S. aureus pathogenesis, deletion of PKM2, or its 357 pharmacological inhibition, results in decreased killing of S. aureus in vitro as a consequence of 358 ablated ROS production (70). Furthermore, in vivo, Pkm2 deficient mice infected with S. aureus 359 show increased bacterial loads and delayed wound healing at the site of infection (70). Products 360 of glycolysis regulated by PKM2, namely diacylglycerol or DAG, will go on to activate PKC -alpha 361 in a calcium dependent fashion (70). This isozyme then phosphorylates the p47phox subunit of the 362 NADPH complex, in turn leading to its activation and ROS production ( 70-71,73). V8 was found 363 to cleave both PKC -alpha and p47 phox at position 283 and position 33, respectively. While PKC -364 alpha is cleaved by V8 directly upstream of its calcium binding C2 domain , p47phox is cleaved in 365 the middle of i ts PX (phox) domain (74,75). The PX domain of the p47 phox subunit of NADPH 366 oxidase, once phosphorylated, mediates the recruitment of this subunit from the cytosol to the 367 phagosome membrane via binding to acidic phospholipids (75). Simultaneously, V8 substrate 368 Rac2 will become activated via GDP to GTP conversion, translocate to the membrane and interact 369 with the NADPH subunits, forming a fully active phagocyte oxidase complex ( 6). To further 370 emphasize the importance of Rac2, studies have shown that activation of the NADPH complex 371 after FcyR mediated phagocytosis is completely dependent on this protein in murine neutrophils 372 (76). As mentioned, Rac2 is a multifunctional protein also displaying a role in leukocyte migration, 373 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint however in this regard, t argeting of Rac2 by V8 could promote S. aureus pathogenesis as a 374 consequence of abrogated ROS production. This is supported by evidence showing patients with 375 decreased levels of this protein experience an autosomal dominant immunodeficiency syndrome 376 characterized by defects in overall neutrophil functionality (72,77). This syndrome is seen mainly 377 in infants where their neutrophils have decreased ROS levels, dampened chemotaxis, 378 polarization and azurophilic granule secretion ( 77). Moreover, patients with Rac2 deficiency 379 present with severe and recurring bacterial infections, accompanied with poor wound healing 380 similar not only to LAD-1, but chronic granulomatous disease (CGD) (70,77-78). 381 Further evidence suggesting V8 is involved in abrogation of ROS production comes from 382 our observed cleavage of the calcium binding chaperone calreticulin. This versatile protein, found 383 to reside not only in lumen of the endoplasmic reticulum, but also on the neutrophil cell surface, 384 has emerged as an important immune response regulator involved in ROS production and 385 pathogen clearance (79 -81). In support of this , previous work investigating the activity of a 386 synthetic antimicrobial peptide demonstrated its ability to bind calreticulin on the surface of 387 neutrophils and induce neutrophil activation and enhance superoxide production via GPCR 388 signaling (80). Consequently, t his peptide exhibited significant anti -staphylococcal action in 389 MRSA infected mice (82). While this AMP may not be in circulation in the normal host 390 environment, this previous study presents evidence for calreticulin as a binding target to induce 391 neutrophil activation, a phenomenon that can be potentially dampened due to V8 cleavage. 392 Calreticulin possess three important domains including the N, P and C -domain, with the former 393 two involved in chaperone function and the C -domain involved in calcium binding (79 -81). We 394 show V8 cleavage of this protein after glutamic acid residues at positions 128 and 342 (Table S2), 395 corresponding to both the N- and C-domains, likely abrogating their function. 396 Intriguingly, additional studies on this molecule have highlighted its pivotal role in protein 397 synthesis and maturation of the ROS mediator, myeloperoxidase, emphasizing the paramountcy 398 of calreticulin in the innate immune response (79). Proposed to stabilize the immature form of 399 MPO to facilitate heme binding (79), cleavage of calreticulin by V8 point s would engender 400 significant dysregulation of ROS production . As such , our data establishes multiple routes by 401 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint which V8 can extinguish intracellular neutrophil defense mechanisms , leading to prolonged 402 survival and persistence within the host. 403 In support of this, we validated V8 cleavage of ROS mediator, pyruvate kinase PKM2. This 404 protein was found to be cleaved t hree times by V8, in t he barrel domain at positions 355 and 405 283, with the latter being just upstream of the active site or bait region, and the C-terminus at 406 position 481 (Figure 4 D). This observed cleavage pattern could give rise to four different 407 degradation products as outlined in the protein schematic ( Figure 4 D). While we observed a 408 decrease in intensity of the full length PKM2 protein and modified form in the V8 treated lanes, 409 we also see an additional band between ~25-35kDa, that corresponds to the molecular weight of 410 cleavage fragments predicted by our N-terminomic data (Figure 4D). 411 412 N-terminomics predicts V8 modulation of phagocytosis: Our study also recorded V8 cleavage of 413 many important neutrophil structural proteins, including myosin-9, tubulin chains, filamin A, and 414 actin subunits. These cytoskeletal proteins are responsible for maintaining important neutrophil 415 functions and characteristics , such as motility, cell shape, membrane organization, and 416 phagocytosis (83,84). Previous work has shown that S. aureus can prevent phagocytosis via anti-417 opsonic strategies, such as cleavage of complement C3 by aureolysin, or via the binding of 418 immunoglobulins by either staphylococcal protein A (Spa) or staphylococcal binding of 419 immunoglobulins (Sbi) protein (3). Herein, our data provides an additional route for how S. 420 aureus can modulate this host defense tactic facilitating immune evasion. Defects in phagosomal 421 cup formation and thus phagocytosis could arise from V8 cleavage of myosin -9, or non-muscle 422 myosin heavy chain IIA, an essential motor protein driving actin polymerization and subsequent 423 extension of neutrophil pseudopod protrusions (84). This notion is supported by a study showing 424 that in the presence of blebbistatin, a specific inhibitor of myosin II, neutrophil phagocytosis is 425 abrogated (85). Given that V8 also cleaves various actin proteins, this suggests dysregulation of 426 the entire process of phagocytosis. Furthermore, as pseudopod extension and actin assembly 427 also propel neutrophils during migration, V8 proteolysis of these proteins provide an additional 428 layer of evidence suggesting that this protease can impede neutrophil migration. Once again, this 429 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint is confirmed by previous work that highlights how application of a myosin IIA inhibitor limits not 430 only phagocytosis but neutrophil migration (85). 431 We took a different approach to validation, whereby we subjected purified human beta 432 actin protein to digestion by V8, with the resulting banding patterns visualized by Coomassie blue 433 staining (Figure 5A). Our N -terminomic data captured cleavage of this molecule at positions 5, 434 148, 227 and 317. The purified protein used was a His-tagged truncated variant with a molecular 435 weight of ~35kDa; the predicted banding patterns and their molecular weights are outlined in 436 the protein schematic. Following V8 proteolysis, we observed two fragments at ~12kDa and 437 ~15kDa, directly corresponding to our N-terminomic results. 438 439 V8 degrades apoptotic signaling proteins: TAGS-CR captured V8 cleavage of pro -apoptotic 440 proteins, including the BH3-interacting domain death agonist (BID) and apoptosis regulator BAK1, 441 and its homolog BAX. These V8 targets are all pro -apoptotic members of the Bcl -2 family that 442 together are essential gateway molecules for inducing apoptosis ( 86). BH-3 only proteins, such 443 as BID, BIM, BAD and PUMA, drive the apoptotic cascade as they induce oligomerization of 444 BAK1/BAX, resulting in mitochondrial outer membrane permeabilization (MOMP), followed by 445 the release of cytochrome c and subsequent activation of downstream caspases, which function 446 as major mediators of programmed cell death ( 86). In healthy cells, the response to apoptotic 447 stress begins with cleavage of BID by caspase-8 at position 59 to form a truncated version of the 448 molecule, tBID, known to induce BAK1/BAX association and subsequent MOMP formation ( 87). 449 Given this, our data points to V8 behaving as a potential activator of the apoptotic process, due 450 to V8 cleavage of BID at position 53, potentially mimicking the caspase-8 effect. 451 Beyond BID, the apoptotic cascade remains complex as the mechanisms of BAK1/BAX 452 activation are not well understood ( 88). However, these molecules have been found to 453 necessitate structural reconfiguration prior to pore formation, including N -terminal 454 conformation changes (89-91). As such, previous work has shown that dissociation of the BAK1 455 α1 helix is necessary for unfolding and exposure of its hydrophobic binding pocket between α2- 456 α5, which mediates interaction with BH-3 only activators such as tBID ( 92). From our data V8 457 cleaves BAK at position 60, found in the α1-α2 loop, which would result in release of the N -458 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint terminal segment and α1 helix (92). Additional studies have explored influence of the α1-α2 loop 459 on BAK 1 activity and determined that it elicits a suppressive effect on BAK 1 activation via 460 stabilization of the molecule ( 93). Interestingly, it was found that amino acid M60 in the α1-α2 461 loop was responsible for stabilization effect of this structure, ultimately preventing downstream 462 activation and subsequent apoptotic pore formation ( 93). Taken together, V8 cleavage within 463 the α1-α2 loop at position 60 indicates that this protease may trigger activation of the apoptotic 464 cascade. 465 While structural rearrangements in BAK1 are akin to those in BAX, it has been 466 hypothesized that α1 dissociation is also necessary for BAX activation ( 92). Herein, we see that 467 V8 may also elicit a similar effect on BAX, as observed for BAK1, cleaving at position 45 in the 468 same structural domain, the α1-α2 loop (95). It is well documented that S. aureus can strategically 469 manipulate host apoptotic processes to permit survival and persistence within the human host. 470 Promotion of apoptosis by virulence factors, such as α-toxin or the pore-forming leukocidins, has 471 been shown to not only induce tissue injury and exacerbate inflammation, but allow for escape 472 and spread to new host niches (96). Given that the secreted proteases have been recognized for 473 their role in promoting dissemination during infection, our study highlights the possible 474 contributory effect of V8 to this phenomenon via cleavage of BID, BAK1 and BAX. 475 As evidence for this claim, we validated cleavage of BAK1. Recombinant GST tagged BAK1 476 was digested with purified V8 , however there was a discrepancy between predicted and 477 corresponding molecular weights of the resulting degradation products. Therefore, we excised 478 the three bands in line with the blue arrow across the different V8 treatment conditions ( Figure 479 5B) and subjected them to in -gel tryptic digest and subsequent MS/MS analysis. In addition to 480 possible self -degradation of the V8 enzyme, the 26kDa GST tag may also have undergone 481 digestion by this protease, therefore we included both protein sequences for spectral assignment 482 when searching the raw data. We considered peptides identified in at least two out of the three 483 treatment conditions as well as having the presence of Glu or Asp residues at either the N- or C-484 terminus, as constituting V8 semi -tryptic peptides ( Table S4). From this we captured 5 reliably 485 detected peptides, 4 identified as the GST tag and 1 corresponding to the BAK1 protein with a 486 start position of 60 as captured by TAGS-CR. 487 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint The V8 protease facilitates survival of S. aureus within human leukocytes: Given that our N -488 terminomic data suggests clear disruption of proper neutrophil functioning by the V8 protease , 489 we sought to assess whether this enzyme contributes to int racellular survival of S. aureus in 490 human neutrophils. As such , a S. aureus ∆sspA mutant was compared to the USA300 LAC wild 491 type for viability in a model of neutrophil survival. At 24h post-infection we determined a clear 492 decrease in the survival of the mutant strain, with bacterial burden decreased around two-fold 493 in comparison to WT (Figure 6). As such, loss of the V8 protease negatively impacts survival of S. 494 aureus inside the phagolysosome of human leukocytes as would be expected from our N -495 terminomic data. Of note, a complementing strain was also included in this experimental model 496 (data not shown), however it failed to complement due to instability of the plasmid; something 497 previously noted in other studies (97-99). 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint SUMMARY AND CONCLUSION 517 The function of proteases as enzymes is defined by their action on other proteins – thus, 518 to understand the role of a protease, one must identify its substrates (25-33, 101). A common 519 misconception is that proteolysis leads to complete degradation. Instead, proteases can cleave 520 with exquisite specificity (particularly V8), rather than randomly, recognizing substrates via key 521 motifs (37, 102-107). Our ability to capture such events has been slow as conventional strategies 522 to assess proteolysis are low throughput, and involve one at a time approaches. However, recent 523 advances in mass spectrometry have led to the field of degradomics, allowing us to capture 524 proteolysis on a global scale (33-36). Herein, we present a novel N-terminomic approach – TAGS-525 CR - that facilitates the rapid, efficient and streamlined resolution of proteolytic events in 526 complex samples. 527 As neutrophils remain steadfast as the front-line defense against invading S. aureus, it is 528 pivotal to understand the molecular mechanisms employed by this pathogen to thwart immune 529 responses and foster infection (52). Using TAGS-CR we determined that V8 can potentially 530 dysregulate a wide breath of neutrophil functions via targeted cleavage of ~350 host proteins. 531 Specifically, we captured proteolysis of critical factors involved in neutrophil adhesion and 532 migration, a fundamental process necessary for pathogen clearance and infection resolution (22). 533 We also showed that V8 can disrupt phagolysosomal defense and apoptosis serving to facilitate 534 S. aureus survival and persistence within the host (12,22,96). Indeed, in the absence of sspA (the 535 V8 encoding gene), S. aureus has an impaired ability to survive during engagement with human 536 neutrophils. 537 We acknowledge that our experimental design may have perceived limitations in the form 538 of access of protease to substrate, facilitated by our use of whole, lysed neutrophil proteomes. 539 We counter this contention with the notion that S. aureus is well adapted to intracellular survival 540 within neutrophils, not only escaping the lethal effects of the phagolyso some, but also thriving 541 within the cytoplasm of these immune cells (108). As such, S. aureus and its proteases would 542 readily have access to most if not all intracellular machinery within neutrophils. In addition, the 543 secreted proteases function in concert with other secreted virulence factors of S. aureus (26). 544 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint Consequently, lytic action on neutrophils by the leukocidins would again readily provide access 545 for the proteases to intracellular components of these immune cells. 546 As such, we suggest that our work illustrates application of a unique N -terminomic 547 strategy and demonstrate s its potential in providing unparalleled insight into S. aureus host-548 pathogen interaction. We present the detailed mapping of the human neutrophil 549 pathodegradome, which not only propels bacterial protease substrate identification but shines 550 new light on the immunomodulatory mechanisms implemented by S. aureus. Furthermore, as 551 neutrophil dysfunction often results in recurrent S. aureus infections, this further emphasizes the 552 significance of our results and offers insight that could facilitate the development of effective 553 treatments and preventative strategies. 554 555

This study was supported by grants AI124458 and AI157506 (L.N.S.) from the 556 National Institute of Allergy and Infectious Diseases. 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint

574 575 Neutrophil Proteome V8 treatmen t: Six flasks of human HL-60 leukemia cells were grown at 37°C with 576 5% CO2 in RPMI supplemented with 10% fetal bovine serum (FBS) and penicillin and streptomycin . HL-60 577 cells were differentiated into neutrophil -like cells via stimulation with 1.25% DMSO. Differentiation was 578 allowed to proceed for 5 days and then cells were centrifuged at 1,000g for 5 minutes. Cell pellets were 579 washed twice with PBS prior to protein extraction. A 50µl portion of each wet cell pellet was resuspended 580 in 500µl of the Mammalian Protein Extraction Reagent M -Per (Thermofisher Scientific) . Proteins were 581 extracted by vigorous shaking for 20 minutes , pooled and then quantified using a Pierce 660nm protein 582 assay (ThermoFisher Scientific). Neutrophil proteomes were standardized to 1mg/ml and incubated 583 ±200ng of V8 protease (MilliporeSigma), at 3 7°C for 16h. Following incubation, protease activity was 584 quenched with dry guanidium hydrochloride (6M final concentration) prior to N-terminomic 585 experimentation. 586 587 N-terminomics: Terminal Amine Guanidination of Substrates (TAGS): Following protease treatment, 588 samples were incubated with 20mM DTT at 95°C for 10 minutes and centrifuged at 17 ,0000g to remove 589 remaining non -solubilized proteins. Iodoacetamide was added to a final concentration of 40mM and 590 incubated in the dark at RT for 30 minutes. Alkylation was quenched with 40mM DTT. Guanidination of 591 protein N-termini was performed by incubating samples with 500mM of triethyl ammonium bicarbonate 592 (TEAB) and 500mM of 1H-pyrazole-1-carboxamidine hydrochloride (HPCA) at 95 °C for 10 minutes. 593 Samples were then quickly chilled on ice for 5 minutes. In accordance with the S-trap protocol (PROTIFI), 594 phosphoric acid was added to a final concentration of 1.2 %. In a 1:7 volumetric ratio, S-trap buffer (10% 595 v/v TEAB pH 7.5, 90% v/v MeOH) was mixed with each sample. Proteins were then bound to the S -trap 596 midi column matrix by repeated flow through at 4 ,000g for 1 minute. Samples were washed a total of 597 three times, each by adding 3ml S-trap buffer and centrifuging as above. For protein digestion, Trypsin/P 598 was added at 1:100 enzyme:protein (10µg of trypsin in 50mM TEAB pH 8.1) and samples were incubated 599 at 37°C for 18 h. Elution of peptides was performed according to the S -trap protocol, with a series of 600 centrifugations starting with 50mM TEAB pH 8.1, then 0.2% (v/v) formic acid and finally with 50% 601 acetonitrile. In a vacuum centrifuge, samples were evaporated to ~1/3 of their final volume. A three-step 602 incubation process was then performed to sulfonate peptides to facilitate downstream enrichment (Lai 603 et al., 2015). Samples were first incubated with 100mM HEPES pH 7.5 and 20mM FBDA and 20mM SCBH 604 for 1h at RT. This was followed by 2 additional incubations with 20mM FBDA and 20mM SCBH at RT, the 605 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint first for 1h and then for 16h. FBDA and SCBH were made fresh prior to each incubation, with the final 606 concentration in the reaction being 60mM FBDA and 60mM SCBH. Following final incubation, excess FBDA 607 and SCBH was quenched by the addition of 2M Tris (pH 8) to a final concentration of 100mM. Using Sep 608 Pak C18 columns (Waters), samples were desalted according to the manufacturer’s protocol. Using a 609 vacuum centrifuge, samples were dried to completion. Strong cation exchange was performed using mini 610 pierce strong cation exchange spin columns (ThermoFisher Scientific). Briefly, peptides were resuspended 611 in 300 µl of SCX buffer A (5mM Ammonium Formate, pH 3.0, 25% (v/v) CAN) and then vortexed and 612 sonicated for 5 minutes each. SCX columns were equilibrated by centrifuging 400 µl of SCX buffer A for 5 613 minutes at 2 ,000g (all SCX centrifugations took place using these parameters unless stated otherwise). 614 Samples were then added to the SCX column and centrifuged, followed by two washes with the addition 615 of 400µl of SCX buffer A and centrifugation. Peptides were eluted from the SCX column with the addition 616 of 200µl of SCX buffer B (500mM Ammonium Formate, pH 6.0, 25% (v/v) ACN) followed by a 1-minute 617 incubation and then centrifugation. This was repeated once to bring the final volume to 400ul. 618 Accordingly, to reduce the ACN to 7.25%(v/v), 1.2mL HPLC water was added to each tube. Samples were 619 acidified with the addition of 80µl of trifluoroacetic acid and an additional desalt was performed using Sep 620 Pak C18 columns (Waters) according to manufacture r’s instructions. Finally, peptides were dried to 621 completion using a vacuum centrifuge and stored at 4°C prior to mass spectrometry. 622 623 Mass Spectrometry: The N-terminally enriched peptides obtained following TAGS -CR were resuspended 624 in 0.1% formic acid. Aliquots of 5µl were separated on a 50cm Acclaim PepMap 100 C18 reversed-phase 625 high-pressure liquid chromatography (HPLC) column (Thermo Fisher Scientific) using an Ultimate3000 626 UHPLC (Thermo Fisher Scientific) with a 120 min gradient (2%-32% aceto-nitrile with 0.1% formic acid). A 627 hybrid Quadrupole -Orbitrap instrument (Q Exactive Plus; Thermo Fisher Scientific) analyzed peptides 628 using data dependent acquisition, with the top 10 most abundant ions being selected for MS2 analysis. 629 630 Data Analysis: Raw files were processed using MaxQuant ( 100). The andromeda search engine included 631 in this program was used to assign MS2 spectra to the reference human proteome (UniProt ID 632 UP000005640). For this N -terminomics experiment, a semi specific digestion setting with trypsin/P was 633 used. Fixed modifications included Carbamidomethyl (C), Guanidination (K), while variable modifications 634 included Oxidation (M), Acetyl (Protein N -term) and Guanidination (N -term). Each experiment was 635 injected into the mass spectrometer twice to give n=6 for both control and V8 treated conditions and 636 labelled appropriately in the MaxQuant software. The match between runs feature was also selected and 637 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint peptide and protein FDR were both set to 1%. The resulting modified peptide output file was analyzed 638 using R programming language whereby contaminants and unmodified peptides were removed and 639 peptides considered for analysis were identified in at least two of the three separately conducted 640 experiments. Subset lists of peptides, proteins, N-termini, and cleavage sites were generated to allow for 641 a binary comparison of unique cleavage events between ±V8 conditions. 642 643 SDS-PAGE and Immunoblotting: SDS-PAGE was performed u sing either 12% or 15% self-cast gels, or 4-644 20% gradient gels (BioRad). Coomassie blue was used to stain gels with recombinant proteins , while gels 645 for digested neutrophil proteomes were immediately transferred to a PVDF membrane at 20 Volts for 45 646 minutes. Following transfer, membranes were blocked (5% milk in Tris buffered saline–Tween 20) for 1h 647 at RT with rocking. Membranes were then exposed to primary antibody (1:1300 – 1:1000 dilution of 648 antibody to blocking buffer) for 16 hours at 4°C and washed 3 times for 10 minutes with blocking buffer. 649 Blots were exposed to the secondary antibody anti-rabbit IgG-HRP conjugate (Cell Signaling Technologies) 650 at a 1:5000 dilution of antibody to blocking buffer for 1h at RT with rocking, and then washed 3 times for 651 10 minutes with blocking buffer. The Supersignal West Pico chemiluminescent substrate (Thermo Fisher) 652 was added to the blot prior to imaging. Blots were captured using developing film having been exposed 653 from 15 minutes to 18h. Primary antibodies used included: Integrin alpha-L (abcam, ab52895), Kindlin-3 654 (Thermo Fisher Scientific, #PA5-30847), Myeloperoxidase (abcam, ab134132), Pyruvate kinase PKM2 655 (Thermo Fisher Scientific, # PA5-28700). Purified recombinant proteins used included: Human beta actin 656 (abcam, ab240844) and Human BAK1 (Abnova, 89-936-369). 657 658 In-Gel Digestion: Designated V8 treated BAK1 bands were excised and cut into 1mmx1mm cubes. Samples 659 were washed twice with 200µl of 50/50 ACN/TEAB by vortexing for 15 minutes, discarding the wash each 660 time. Gel pieces were then dehydrated by covering with ACN for 10 minutes. Once ACN was removed, 661 samples were rehydrated with 50µl of 100mM TEAB and left to incubate for 5 minutes. To each sample, 662 an equal volume of ACN was then added to achieve a 1:1 ratio of ACN:100mM TEAB and a final wash was 663 performed. Following removal of the wash, samples were dried in a vacuum centrifuge for 5 minutes and 664 then incubated at 55°C for 30 minutes in the presence of 100µl of 50mM DTT. Once samples were allowed 665 to return to RT, they were then incubated in 100µl of 100mM IAA in the dark at RT. IAA was removed and 666 gel pieces were washed three times as described above and then dried for 5 minutes in a vacuum 667 centrifuge. To each sample, 100ng of trypsin was added, followed by overnight incubation at 37 °C. 668 Peptides were collected by transferring the supernatant to a fresh microcentrifuge tube and by 669 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint performing two subsequent washes, adding each to the new designated sample tube. Once peptides were 670 dried to completion in a vacuum centrifuge, m ass spectrometry was performed as described above and 671 raw files were processed using MaxQuant (100). The resulting peptide file was analyzed and 672 contaminants, peptides not found in at least 2/3 BAK1 V8 treated lanes, as well as non -V8 semi-tryptic 673 peptides, were removed. 674 675 Data Deposition: Raw mass spectrometry data for this study was deposited as two submissions to the 676 ProteomeXchange Consortium using the PRIDE repository with the dataset IDs PXD057551 and 677 PXD057579 for the neutrophil N-terminomics and the BAK1 in-gel digestion, respectively. 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint

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The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint 108. Fraunholz, M. and Sinha, B., 2012. Intracellular Staphylococcus aureus: live -in and let 1012 die. Frontiers in cellular and infection microbiology, 2, p.43. 1013 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint Figure 1. Guanidination and Enrichment of N-terminal peptides through TAGS-CR. Proteins are reduced and cysteine residues are blocked via alkylation prior to guanidination of N-terminal α-amines and lysine ε-amines (not shown) using 1H-Pyrazole-1-carboxamidine (HPCA). A manufacturer provided S-trap protocol is used and N-terminally labelled proteins are tryptically digested. Following elution, peptides undergo (di)sulfonation of free amines that have been liberated post tryptic digest. Strong cation exchange (SCX) is then performed to enrich for both native and neo-N-termini, the latter of which indicates a cleavage event and thus only pertains to the protease treated condition. LC-MS/MS analysis is then performed on the TAGS-CR enriched samples to obtain the N-terminome. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint (A) (B) P5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ Figure 2. TAGS-CR validates cleavage specificity of the V8 protease. Analysis of the amino acid sequence surrounding the site of proteolysis provided immediate validation of V8 induced cleavage of our 346 neutrophil targets by showcasing the known precision of this enzyme to cleave primarily at the carboxyl side of glutamic acid (E) residues. Demonstrating this, ~90% of residues upstream of the cleavage site accounted for glutamic acid (E) residues (A). We performed an additional dimension of analysis using ICELOGO whereby we looked at the percentage difference of significantly increased and decreased residues at the five positions (P5-P5’) surrounding the sessile bond. Here we found that only glutamic acid was significantly increased at P1 or upstream of the cleavage site (B). (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint Figure 3. V8 targets multiple dimensions of neutrophil functionality and defense relevant to S. aureus pathogenesis. Green molecules are highlighted as key neutrophil proteins cleaved by V8. Displayed adjacent to these are the corresponding neutrophil processes they play a role in. These include: (A) Leukocyte Extravasation, (B) Integrin Interactions, (C) Neutrophil Degranulation, (D) ROS Production, (E) Apoptosis, and (F)Phagocytosis. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint Integrin alpha-L 10- 15- 25- 35- 55- 70- 100- 250- 130- KEY: C: Control T: V8 Treated kDa C1 T1 T2 T3 (A) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint Kindlin-3 KEY: C: Control T: V8 Treated kDa 10- 15- 25- 35- 55- 70- 100- 250- 130- C1 T1 T2 T3 (B) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint Myeloperoxidase 10- 15- 25- 35- 55- 70- 100- 250- 130- C1 T1 T2 T3 KEY: C: Control T: V8 Treated kDa 6 (C) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint PKM2 kDa KEY: C: Control T: V8 Treated 10- 15- 25- 35- 55- 70- 100- 250- 130- C1 T1 T2 T3 (D) Figure 4. Immunoblot validation of V8 neutrophil targets. Neutrophil proteomes were exposed to 200ng of the V8 protease for 16h at 37°C whilst control conditions remained untreated. Degradation patterns and their corresponding approximate molecular weights (calculated using Bioinformatics.org) according to the cleavage events captured by our N-terminomic data are outlined in the relevant protein schematic. Also highlighted in the schematics are the identified sites of V8 cleavage. Western blot analysis of neutrophil V8 targets include: (A) Integrin alpha-L, (B) Kindlin, (C) Myeloperoxidase, and (D) Pyruvate kinase PKM2. VWFA = von Willebrand A. FERM = Ferm domain. PH = PH domain. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint 10- 15- 25- 35- 55- 70- 100- 250- 130- kDa Human Beta Actin (A) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint 10- 15- 25- 35- 55- 70- 100- 250- 130- kDa BAK1 Figure 5. SDS-Page validation of neutrophil V8 targets using purified recombinant proteins. Recombinant proteins of (A) Human beta actin and (B) BAK1 were treated with varying concentrations of the V8 protease and compared to untreated controls. Purified V8 was also included as a control. A schematic of the corresponding protein is presented adjacent to the Coomassie Blue stained gel. Green coloring indicates the corresponding portion of the protein that was purchased. Cleavage sites detected by TAGS-CR are highlighted with the asterisk and the surrounding residues, as well as the resulting degradation products and their corresponding molecular weights. The blue arrow indicates bands that were excised for in-gel digest for (B) BAK1. (B) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint Figure 6. The V8 protease promotes intracellular survival of S. aureus within human leukocytes. HL-60 derived human neutrophils were infected with either the S. aureus LAC wild type or the sspA (V8 encoding gene) mutant strain using an MOI of 13.5. CFU/mL was then determined after a 24-hour period and data displayed represents the average of 3 biological replicates for each strain. The significance of relative bacterial burden between strains was determined using an unpaired t-test with unequal variance, **p=<0.01. Error bars are ± SEM. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 8, 2024. ; https://doi.org/10.1101/2024.11.08.622692doi: bioRxiv preprint