Neutrophils use selective autophagy receptor p62/SQSTM1 to target Staphylococcus aureus for degradation in vivo in zebrafish

Autophagy leads to degradation of cellular components and has an important role in restricting intracellular pathogens. Autophagy receptors, including p62, target invading intracellular pathogens to the autophagy pathway for degradation. Staphylococcus aureus is a significant pathogen of humans and often life-threatening in the immunocompromised. Increasing evidence demonstrates that S. aureus is an intracellular pathogen of immune cells and may use neutrophils as proliferative niche but the intracellular fate of S. aureus following phagocytosis by neutrophils has not previously been analysed in vivo. In vitro, p62 is able to co-localise with intracellular Staphylococcus aureus, but whether p62 is beneficial or detrimental in host defence against S. aureus in vivo had not been determined. Here we use zebrafish to determine the fate and location of S. aureus within neutrophils throughout infection. We show that Lc3 and p62 recruitment to phagocytosed S. aureus is altered depending on the bacterial location within the neutrophil. We also show rapid Lc3 marking of bacterial phagosomes within neutrophils which may be associated with subsequent bacterial degradation. Finally, we find that p62 is important for controlling cytosolic bacteria demonstrating for the first time a key role of p62 in autophagic control of S. aureus in neutrophils.

Abstract 29 30 Autophagy leads to degradation of cellular components and has an important role in restricting 31 intracellular pathogens. Autophagy receptors, including p62, target invading intracellular 32 pathogens to the autophagy pathway for degradation. Staphylococcus aureus is a significant 33 pathogen of humans and often life-threatening in the immunocompromised. Increasing 34 evidence demonstrates that S. aureus is an intracellular pathogen of immune cells and may 35 use neutrophils as proliferative niche but the intracellular fate of S. aureus following 36 phagocytosis by neutrophils has not previously been analysed in vivo. In vitro, p62 is able to 37 co-localise with intracellular Staphylococcus aureus, but whether p62 is beneficial or 38 detrimental in host defence against S. aureus in vivo had not been determined. 39 Here we use zebrafish to determine the fate and location of S. aureus within neutrophils 40 throughout infection. We show that Lc3 and p62 recruitment to phagocytosed S. aureus is 41 altered depending on the bacterial location within the neutrophil. We also show rapid Lc3 42 Autophagy (macroautophagy) is a process of cellular self-degradation, in which damaged or 50 redundant cellular components are taken into an autophagosome and subsequently trafficked 51 to the lysosome for degradation; these degraded components can then be recycled for 52 alternative uses by the cell (Mizushima et al., 2008;Tanida, 2011). During infection, autophagy 53 is used by host cells to degrade invading pathogens, a process termed xenophagy (Gatica,54 Lahiri and Klionsky, 2018; Sharma et al., 2018). 55 56 Autophagy is considered largely non-selective of the cargo to be degraded, classically being 57 induced by starvation conditions. However, selective autophagy is a process that enables 58 specific cargo to be directed into the autophagy pathway, which can be used to target invading 59 pathogens. Selective autophagy uses autophagy receptors (ARs), proteins that interact with 60 both autophagy machinery and the cargo to be degraded (Popovic and Dikic, 2012;Rogov et 61 al., 2014). Many ARs are involved in targeting invading pathogens, including p62 (also named 62 sequestosome 1 (SQSTM1)), neighbour of Brca1 gene (NBR1), optineurin (OPTN) and 63 nuclear dot protein 52 (NDP52) (Farré and Subramani, 2016). 2019) can be targeted by ARs for degradation. Conversely, pathogens have evolved to be 71 able to block or subvert immune defences, and autophagy is no exception. Indeed, many 72 bacterial pathogens are able to inhibit induction of autophagy or to reside within the autophagy 73 pathway by preventing lysosomal fusion, or even avoid making any contact with autophagic 74 machinery (Deretic and Levine, 2009 (Schnaith et al., 2007) or detrimental for S. aureus (Neumann et al., 2016). 84 Intracellular pathogens, including S. aureus, are able to escape the phagosome into the 85 cytosol (Bayles et al., 1998), likely through toxins secreted by the bacteria or membrane 86 rupture due to bacterial growth. Once in the cytosol, bacteria can be ubiquitinated and targeted 87 by ARs (Farré and Subramani, 2016). Indeed, p62 in fibroblasts and epithelial cells has been 88 shown to localise to cytosolic S. aureus leading to autophagsome formation in vitro (Neumann 89 et al., 2016;Singh et al., 2017). Therefore, we investigated whether p62 recruitment is 90 in infection. Therefore, to determine the fate and location of S. aureus in neutrophils during 106 infection, S. aureus expressing mCherry was inoculated and imaged at early (2 to 5 hours 107 post infection (hpi)) and late (24 to 28hpi) time points. Initially, the well-established 108 Tg(mpx:eGFP)i114 line that specifically marks neutrophils with EGFP (Renshaw et al., 2006) Figure 1A). 117 We next sought to determine the location of bacteria, and relationship to autophagic 118 machinery, within neutrophils. To do this we used the newly generated Tg(lyz:RFP-GFP-119 Lc3)sh383 (Prajsnar et al., 2019). We first confirmed that, in the caudal hematopoietic tissue 120 (CHT), the infection dynamics were similar to the Tg(mpx:eGFP)i114 line, with a significant 121 reduction in intracellular bacteria by 26hpi, indicating bacteria are efficiently controlled and a 122 significant reduction in infected neutrophils was observed ( Figure 1D). Importantly, the number 123 of neutrophils analysed in the CHT, used for analysis throughout this study, did not significantly 124 change between 2dpf and 3dpf (Supplementary Figure 1B), demonstrating that the change in 125 proportions of infected neutrophils is not due to a large increase in neutrophil number between 126 these time points. The labelling of S. aureus containing vesicles enabled the identification of 127 intracellular bacteria that were within a vesicle ( Figure 1E) or free in the cytosol ( Figure 1F), 128 as well as non-labelled vesicles, or vesicles marked with Lc3 puncta (Supplementary Figure  129 1C, D). We found that the proportion of bacteria within vesicles was significantly reduced over 130 time post-injection, whereas the number of bacteria within the cytosol remains relatively 131 constant at a low level, despite becoming proportionally higher relative to vesicular bacteria 132 ( Figure 1G). Thus, S. aureus phagocytosed by a neutrophil are initially located in a phagocytic 133 vesicle and are subsequently degraded. However, a smaller proportion of S. aureus is able to 134 survive to later infection time points, and these predominantly reside in the cytosol. 135

Generation and characterisation of an in vivo neutrophil GFP-p62 reporter line 136
A previous study identified colocalisation of p62 with S. aureus in non-immune cells (Neumann 137 et al., 2016). Our findings demonstrated a small but significant population of bacteria that were 138 cytosolic, and therefore a possible target for p62 binding. Accordingly, we generated a 139 transgenic neutrophil-specific p62 reporter zebrafish line to examine whether p62 and 140 intracellular pathogens are co-localised in vivo. We used GFP fused via a small linker region 141 to the N-terminus of p62 in order to produce a fluorescently marked fusion protein expressed 142 within neutrophils via the lysozyme C (lyz) promoter (Yang et al., 2012). Using larvae with 143 double labelled neutrophils, we were able to identify GFP expressing cells from the 144  We next examined whether the GFP-p62 protein is able to function as expected. Interestingly, 148 in the double labelled larvae, GFP puncta but not mCherry puncta were seen (Supplementary 149 Figure 2D). Similar p62 puncta that required UBD to function have been observed in vitro for 150 endogenous p62 (Bjørkøy et al., 2005). To test whether the GFP-p62 puncta observed in the 151 GFP-p62 reporter line respond as expected, GFP-p62 reporter larvae were treated with 152 autophagy inhibitor Bay-K8644 (known to block autophagy in zebrafish; (Williams et al., 2008).  Figure 3B). This suggested that neutrophils 165 were efficiently degrading these bacteria, in agreement with Figure 1C. Having characterised features consistent with a S. aureus containing vacuoles we were able 181 to assign a subset of bacteria as being in either a damaged phagosome or located in the 182 cytosol (p62GFP high , Supplementary Figure 3F); for the purpose of this study we are defining 183 these bacteria as cytosolic as they are accessible to cytosolic proteins. We then assigned the 184 cellular location of S. aureus by these features at 2hpi and 26hpi. We determined that the 185 proportion of S. aureus within vesicles was significantly reduced by 26hpi (Supplementary 186   represent Lc3-associated phagocytosis (LAP), which is also observed in Listeria 299 monocytogenes infection of macrophages (Gluschko et al., 2018). Since most bacteria are 300 degraded, it appears that Lc3 marking of vesicles could lead to bacterial degradation in the 301

zebrafish. 302
Thus, we demonstrate that host p62 is beneficial for the host outcome following S. aureus 303 infection and that p62 mediated control of cytosolic bacteria within neutrophils may represent 304 one of many mechanisms employed by the host in immunity to this versatile pathogen. and Tg(mpx:eGFP)i114 (Renshaw et al., 2006). Generation of p62 sh558 mutant zebrafish is 328 described below. Larvae were maintained in E3 plus methylene blue at 28°C until 5dpf. 329

Zebrafish micro-injection 339
For p62 morpholino micro-injections: Larvae were injected immediately after fertilisation 340 using a p62 morpholino (van der Vaart et al., 2014). A standard control morpholino 341 (Genetools) was used as a negative control. For injection of S. aureus, zebrafish larvae were 342 injected at 1 dpf (for survival analysis, (Prajsnar et al., 2008)) or 2 dpf (for microscopy 343 analysis) and monitored until a maximum of 5dpf. Larvae were anesthetised by immersion 344 in 0.168 mg/mL tricaine in E3 and transferred onto 3% methyl cellulose in E3 for injection. 345 For S. aureus 1nl of bacteria, containing 1500cfu, was injected into the yolk sac circulation 346 valley. Larvae were transferred to fresh E3 to recover from anaesthetic. Any zebrafish 347 injured by the needle/micro-injection were removed from the procedure. Zebrafish were 348 maintained at 28°C.

Microscopy of infected zebrafish 363
Larvae were anaesthetized 0.168 mg/mL tricaine in E3 and mounted in 0.8% low melting 364 agarose onto glass bottom microwell dishes (MatTek P35G-1.5-14C). An UltraVIEW VoX 365 spinning disk confocal microscope (Perkin Elmer, Cambridge, UK) was used for imaging 366 neutrophils within larvae. 405nm, 445nm, 488nm, 514nm, 561nm and 640nm lasers were 367 available for excitation. Most cellular level imaging was completed in the caudal hematopoietic 368 tissue (CHT) using a 40x oil objective (UplanSApo 40x oil (NA 1.3)). In some cases a 20x 369 objective was used for whole larvae imaging. GFP, TxRed emission filters were used and 370 bright field images were acquired using a Hamamatsu C9100-50 EM-CCD camera. Volocity 371 software was used. Between early and late time points zebrafish larvae were placed back into 372 E3 and maintained at 28°C. 373 pHrodo staining of S. aureus 374 Bacterial strains were prepared for injected (as above) and re-suspended into PBS pH 9.

Image analysis 398
Image analysis was performed using ImageJ software, to quantify the number of S. aureus 399 cells within neutrophils, and to quantify GFP-p62 puncta and Lc3 co-localisation to these 400

pathogens. 401
Drug treatment of zebrafish 402 Larvae were treated with autophagy inducers and inhibitors through immersion in E3 medium. 403 All drugs were sourced from Sigma-Aldrich, UK. The Bay K8644 was added to the E3 to the 404 required concentration, Bay-K 6844 1µM. Larvae were incubated at 28°C for 24 hours before 405 microscopy. Zebrafish were not anaesthetised for immersion drug treatments. 406

Generation of p62 mutant 407
A zebrafish p62 mutant was generated using CRISPR/Cas9 mutagenesis. A guide RNA 408 targeting exon 8 of zebrafish p62 (ACAGAGACTCCACCAGCCTA) was inserted into a 409 published oligonucleotide scaffold (Talbot and Amacher, 2014) and injected together with 410 recombinant Cas9 protein (New England Biolabs) into 1-2 cell stage zebrafish (AB strain). 411 Efficiency of mutagenesis was confirmed using high resolution melt curve analysis as 412 previously described (Sutton et al., 2007) and several founders were identified. P62 sh558 413 carries a 10 base pair deletion resulting in a frameshift and premature truncation of p62 in the 414 ubiquitin-associated (UBA) domain. 415

Statistical analysis 416
Statistical analysis was performed as described in the results and figure legends. We used 417 Graph Pad Prism 7 (v7.04) for statistical tests and plots.