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. Author manuscript; available in PMC: 2022 Jan 1.
Published in final edited form as: Methods Mol Biol. 2022;2303:605–625. doi: 10.1007/978-1-0716-1398-6_46

Role of HSPGs in Systemic Bacterial Infections

Rafael S Aquino 1, Kazutaka Hayashida 1, Atsuko Hayashida 1, Pyong Woo Park 1,2
PMCID: PMC8560348  NIHMSID: NIHMS1747638  PMID: 34626410

Abstract

Heparan sulfate proteoglycans (HSPGs) are at the forefront of host–microbe interactions. Cell surface HSPGs are thought to promote infection as attachment and internalization receptors for many bacterial pathogens and as soluble inhibitors of host immunity when released from the cell surface by ectodomain shedding. However, the importance of HSPG–pathogen interactions in vivo has yet to be clearly established. Here we describe several representative methods to study the role of HSPGs in systemic bacterial infections, such as bacteremia and sepsis. The overall experimental strategy is to use mouse models to establish the physiological significance of HSPGs, to determine the identity of HSPGs that specifically promote infection, and to define key structural features of HSPGs that enhance bacterial virulence in systemic infections.

Keywords: Heparan sulfate, Heparin, Proteoglycan, Syndecans, Bacteremia, Sepsis, Host defense

1. Introduction

Sepsis is a systemic infectious disease associated with organ injury, dysfunction, and failure [1]. Mortality associated with sepsis remains high and is over 25% among approximately 750,000 patients annually in the US alone [1–3]. Globally, the yearly incidence of sepsis is estimated at 31 million cases [1]. The cost of sepsis-associated medical care, estimated at $17 billion annually in the US [4], is escalating. Moreover, because sepsis is common in the elderly, the incidence is projected to increase as the population ages [3]. Based on the current paradigm that sepsis is a syndrome where infection instigates disease but the dysregulated host response mediates disease progression [1, 5–7], more than 100 randomized clinical trials have tested if modulating components and pathways of the septic host response to infection can improve survival. With one short-lived exception, none of these has resulted in effective treatments for sepsis [1, 8, 9]. These data clearly indicate an unmet need for therapeutic options in sepsis and suggest that new targets, pathways, and ideas need to be rigorously examined and existing paradigms need to be refined.

Heparan sulfate (HS) and its pharmaceutical analog, heparin, bind and regulate many factors implicated in bacterial pathogenesis and host immunity, such as microbial adhesins and secreted virulence factors, cytokines, chemokines, and antimicrobial peptides [10–14]. However, precisely how HS modulates the pathogenesis of infectious diseases in vivo remains largely unknown. HS is a glycosaminoglycan (GAG) comprised of unbranched, repeating disaccharide units of hexuronic acid, either glucuronic acid or iduronic acid, alternating with an unsubstituted or N-substituted glucosamine on which the substituents are either acetate or sulfate [15, 16]. All HS in vivo is found covalently conjugated to specific core proteins as HSPGs [10, 17]. In HSPG biosynthesis, a non-sulfated HS precursor is polymerized on specific Ser residues of HSPG core proteins and then extensively and variably modified in the Golgi by N-deacetylase N-sulfotransferases (NDSTs) that catalyze the N-deacetylation and N-sulfation of glucosamine, C5 epimerase that catalyze the epimerization of uronic acid, and 2-O-sulfotransferase (2OST), 6OSTs, and 3OSTs that catalyze the O-sulfation of both glucosamine and uronic acid [15, 18]. HSPG biosynthesis is not template-driven, and the modification reactions do not go to completion, resulting in the generation of highly heterogeneous mature HS chains that differ in size, charge density, and degree of sulfation. The unique and complex structural pattern of HS is thought to enable HSPGs to interact specifically with many molecules [19], but the precise contribution of HS modifications in biological processes in vivo is still largely unknown.

Available data suggest that HSPGs promote bacterial infections by serving as an attachment/internalization receptor at the cell surface and as a soluble inhibitor of innate immunity in the extracellular environment when released by ectodomain shedding. Many bacteria bind to the HS moiety of cell surface HSPGs, and these interactions have been shown to facilitate pathogen attachment and invasion of host cells in vitro [11, 20]. For example, binding of Neisseria gonorrhoeae to syndecan-1 (Sdc1) on epithelial cells is thought to trigger signaling that leads to bacterial internalization [21, 22]. However, HSPGs do not always mediate the internalization of HS/heparin-binding pathogens. Staphylococcus aureus, for example, can invade host cells, survive intracellularly, and bind to HS [23, 24], but both clinical isolates and laboratory strains of S. aureus do not bind to Sdc1 and Sdc1 does not support S. aureus adhesion to human and mouse corneal epithelial cells [25]. How HS/heparin-binding pathogens interact selectively with specific HSPGs and why only certain microbial HSPG interactions lead to ligand internalization are not understood. Furthermore, several HS/heparin-binding bacterial pathogens activate Sdc1 shedding and remove Sdc1 from the host cell surface [26–29], clearly indicating that Sdc1 is not the preferred HSPG receptor for their adhesion and internalization. Studies suggest that bacterial pathogens activate Sdc1 shedding to exploit the ability of Sdc1 ectodomains to inhibit innate immune mechanisms and promote their survival in the hostile host environment [25, 26, 30]. For example, infection of mice with either intravenous (i.v.) or intraperitoneal (i.p.) S. aureus strain USA300 activates Sdc1 shedding in the liver and leads to a marked reduction of Sdc1 on the sinusoidal cell surface of hepatocytes (Fig. 1).

Fig. 1.

Fig. 1

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Systemic S. aureus infection induces Sdc1 shedding. Wt mice on the C57BL/6J background were infected i.v. or i.p. with 5 × 107 cfu of S. aureus USA300, and their livers were isolated at 24 h post-infection. Paraffin-embedded liver sections of unchallenged mice and infected mice were immunostained for Sdc1 with Alexa 647-conjugated 281.2 rat anti-mouse Sdc1 monoclonal antibodies (original magnification, ×200)

Here we describe preclinical experimental approaches to study the role of HSPGs in systemic bacterial infections, such as bacteremia and sepsis. Rodents, and in particular, mice, are used frequently in preclinical studies of infectious disease because of their relatively rapid reproduction cycle, small size, ease of handling and housing, relative cost-effectiveness, availability of many inbred strains (e.g., C57BL/6, BALB/c), and the availability of genetically modified mouse lines. Mice are also readily amenable to experimental prophylactic and therapeutic approaches, and their immune system is well characterized. However, it must be noted that therapeutic approaches for sepsis discovered in mice have so far rarely proven to be successful in large clinical studies. Several factors are thought to contribute to difficulties in translating results from preclinical animal studies to clinical sepsis, including the complexity of sepsis pathogenesis, usage of inbred mice, and methods and approaches not fully mimicking the clinical condition, among others. Regardless, it is also true that mouse models have helped investigators identify new molecular targets, define key biological mechanisms, and test the safety and efficacy of potential new therapeutic approaches. Thus, while mouse models to study systemic bacterial infections described below are established, readers should keep in mind that these protocols need to be further refined and revised to better recapitulate the clinical conditions.

2. Materials

2.1. Intravenous and Intraperitoneal Bacterial Infection

  1. Mice: Mice are generally used at the age of 5–12 weeks (see Note 1). Younger and older mice can be used to study age-dependent responses, but very young mice will not be suitable for i.v. bacterial infection because of the size of their veins. Inbred wild-type (Wt) mice are available from several vendors (e.g., Jackson Laboratory, Bar Harbor, ME; Charles River Laboratories, Wilmington, MA; Taconic, Hudson, NY; Harlan Laboratories, Indianapolis, IN) (see Note 2).

  2. Bacterial nutrient broth: Powder stocks and pre-made solutions of trypticase soy broth (TSB) and brain heart infusion (BHI) broth are available from several vendors (e.g., BD Biosciences, Franklin Lakes, NJ; Thermo Fisher Scientific, Waltham, MA; Millipore Sigma, Burlington, MA; VWR, Westchester, PA). When using powder stocks, sterilize resuspended solutions by autoclaving.

  3. Bacterial nutrient agar: Powder stocks and pre-made plates of trypticase soy agar (TSA) and BHI agar are available from several vendors (e.g., BD Biosciences, Franklin Lakes, NJ; Thermo Fisher Scientific, Waltham, MA; Millipore Sigma, Burlington, MA; VWR, Westchester, PA).

  4. S. aureus: Protocols are described for i.v. and i.p. S. aureus infections. Several S. aureus strains are available from ATCC (Manassas, VA) (see Note 3). We generally use the methicillin-resistant S. aureus (MRSA) strain USA300 and the laboratory strains 8325-4, Newman, and Woods for our studies. S. aureus strains can be stored short-term on TSA slants or plates at 4 °C or long-term in 40% glycerol/TSB at −80 °C.

  5. Listeria monocytogenes: Protocols are described for i.v. Listeria monocytogenes infections. Several L. monocytogenes strains are available from ATCC (Manassas, VA) (see Note 3). We generally use strains EGDe and 10403S for our L. monocytogenes studies. L. monocytogenes strains can be stored short-term on BHI agar slants or plates at 4 °C, or long-term in 40% glycerol/BHI broth at −80 °C.

  6. Mouse restraining device: Rodent restrainers of various size are available from several vendors (e.g., Thermo Fisher, VWR).

  7. Micropipettes and tips are available from general supply vendors (e.g., Thermo Fisher; VWR). Sterilize pipette tips by autoclaving.

  8. Sterile syringes and needles (25–30G) are purchased sterile from general supply vendors.

  9. Sterile gauze and alcohol pads are from general vendors.

  10. Tissue straining medium: Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). Do not add antibiotics to the medium (i.e., penicillin and streptomycin). Filter sterilize.

  11. Sterile cell strainer (70 μm mesh size) is from general vendors.

  12. Sterile polystyrene petri dishes (i.e., low-priced ones for pouring bacterial plates) are from general vendors.

  13. Polypropylene microcentrifuge tubes are from general vendors. Sterilize microfuge tubes by autoclaving.

  14. TSB or BHI broth containing 0.1% (v/v) Triton X-100: Add Triton X-100 to autoclaved TSB or BHI broth, mix, and filter sterilize.

  15. Spectrophotometer: A conventional spectrophotometer (Bio-Rad Laboratories, Thermo Fisher Scientific, VWR) and disposable plastic cuvettes (Thermo Fisher Scientific, VWR) are used to measure the turbidity of bacterial suspensions at OD600nm to estimate the concentration.

2.2. Cecal Ligation and Puncture (CLP)-Induced Polymicrobial Sepsis

  1. Mice: Mice are generally used at the age of 5–12 weeks, but younger and older mice could be used to study age-dependent responses (see Note 1). Inbred wild-type (Wt) mice are available from several vendors (e.g., Jackson Laboratory; Charles River Laboratories; Taconic; Harlan Laboratories) (see Note 2).

  2. Ketamine (100–120 mg/kg) and xylazine (5–10 mg/kg) anesthetic.

  3. Fine scissors and forceps are from Fine Science Tools (Foster City, CA) or Roboz (Gaithersburg, MD). Sterilize surgical tools by autoclaving.

  4. Surgical boards are from Fine Science Tools or Roboz.

  5. Sterile silk sutures (4-0) with needle.

  6. Hair clipper.

  7. Sterile eye ointment.

  8. Betadine solution.

  9. Alcohol pads.

  10. Sterile gloves.

  11. Sterile gauze.

  12. Sterile 3 ml syringe with 20G needle.

  13. Heating pad.

  14. Autoclavable surgical drapes.

  15. Weighing scale.

  16. Stainless steel sterilization tray: Place non-sterile surgical tools and materials in a sterilization tray, wrap the tray with surgical drapes, and autoclave.

  17. Pre-warmed sterile saline.

  18. Buprenorphine.

2.3. Heparan Compounds and Heparan Antagonists in Systemic Bacterial Infection

  1. Heparan compounds: Purified HS and heparin (and other GAGs as controls) are available from several commercial sources (e.g., Galen Laboratory Supplies, North Haven, CT; Iduron, Manchester, UK; R&D Systems, Minneapolis, MN; Millipore Sigma) (see Note 4). Make concentrated stock solutions in autoclaved deionized water or neutral buffer (e.g., PBS) and store at 4 °C for short-term storage or at −80 °C for long-term storage.

  2. Heparan antagonists: Many general inhibitors of GAGs are available from commercial sources (e.g., Millipore Sigma). These include the cationic compounds such as protamine and surfen, and polysulfated anionic compounds such as carrageenans and suramin, among others [31, 32]. Make concentrated stock solutions in autoclaved deionized water or neutral buffer and store at 4 °C for short-term storage or at −80 °C for long-term storage.

2.4. Heparin Lyases and Heparan Derivatives in Systemic Bacterial Infection

  1. Heparin lyases: Heparinase I, II, and III (and other GAG lyases as controls) are available from commercial sources (e.g., Galen Laboratory; Millipore Sigma; R&D Systems; Iduron; Amsbio, Lake Forest, CA). Make concentrated stock solutions in autoclaved neutral buffer and store small aliquots at −80 °C. Do not repeat freeze-thaw.

  2. Heparin derivatives: Selectively desulfated heparin compounds, oversulfated heparin, and heparin oligosaccharides are available from commercial sources (e.g., Galen Research Laboratory; Millipore Sigma) (see Note 5). Make concentrated stock solutions in autoclaved deionized water or neutral buffer and store at 4 °C for short-term storage or at −80 °C for long-term storage.

2.5. Genetically Modified Mice in Systemic Bacterial Infection

  1. KO mouse lines: Several KO mouse lines lacking genes for specific HS modification enzymes or HSPG core proteins have been published. For HS modification enzymes, mice lacking Ndst2 [33], Ndst3 [34], Hs6st2 [35], or Hs3st1 [36] are viable. Global ablation of other HS modification enzymes results in either embryonic (e.g., Ext1, HS6st1) or perinatal (e.g., Ndst1, Glce, Hs2st) lethality. For HSPGs, global KO mice lacking Sdc1 [30, 37], Sdc3 [38], Sdc4 [39], glypican-1 (Gpc1) [40], Gpc3 [41], Gpc4 [42], serglycin (Prg1) [43], or collagen XVIII (Col18a1) [44] are viable. Contact the corresponding principal investigator for availability of these mice (see Note 6).

  2. Conditional KO mouse lines: Several conditional KO lines for HS modification enzymes and HSPG core proteins have been published. Mice harboring a floxed construct of Ext1 [45], Ndst1 [46], Hs2st [47], or Hs6st1 [47] have been generated and ablated in various cell types by crossing with cell-specific Cre driver lines. Mice harboring conditional KO constructs for Sdc2 [48] and Gpc6 [49] have also been generated and are viable. The floxed (or FRT) conditional KO mice can be crossed with other Cre (or flippase) driver mice to ablate HS modification enzymes and HSPG core proteins in specific cells or tissues. Contact the corresponding principal investigator for availability of these mice.

3. Methods

We describe here procedures for mouse models of i.v. and i.p. bacterial infection using S. aureus and L. monocytogenes as pathogens, and CLP-induced sepsis, and how to apply these models to study the role of HSPGs in systemic bacterial infections. These general methods can be applied to other bacterial pathogens and to study the role of other proteoglycans and GAGs in bacteremia and sepsis. The i.v. and i.p. models described here use a single species of bacteria and thus cause monomicrobial bacteremia and sepsis. On the other hand, CLP-induced sepsis is polymicrobial and is generally considered as the more physiologically relevant sepsis model. However, the monomicrobial models allow more rigorous investigation of specific host–pathogen interactions and can also be relevant for certain bacterial pathogens that can cause clinically significant systemic infections on their own.

S. aureus is the leading cause of both community-acquired and healthcare-associated bacteremia, with a high mortality rate of about 20% [50]. In fact, S. aureus is the leading cause of human bacterial infections. In the USA, approximately 500,000 individuals are infected by S. aureus annually, and the economic burden of S. aureus infections in 2003 was estimated to be ~$14.5 billion dollars [51, 52]. One particular MRSA strain, USA300, has an enhanced virulence phenotype that enables it to infect otherwise healthy individuals, and USA300 infections are now considered of epidemic proportion in the USA. Figure 2 shows that S. aureus USA300 causes significant infection in wild-type (Wt) mice on the BL/6 J background when injected either i.v. or i.p. Significant USA300 infection is observed in various organs, including the liver, lung, and spleen, consistent with the association of S. aureus bacteremia and sepsis with multi-organ injury (MOI) and multi-organ dysfunction (MOD) (Fig. 2A). Extensive tissue colonization of i.v. or i.p. S. aureus can be visualized by immunostaining tissues sections for poly-N-acetylglucosamine (PNAG), a cell surface polysaccharide expressed by many bacterial pathogens, including S. aureus, L. monocytogenes, and Streptococcus pneumoniae [53] (Fig. 2b). Host defense against S. aureus infections is primarily mediated by neutrophils and the most common pathogen in patients with neutrophil deficiencies is S. aureus [54]. Consistent with these features of S. aureus infections, a large number of neutrophils are recruited to infected tissues in mouse models of i.v. and i.p. S. aureus infection, which can be visualized by immunostaining tissue sections for the neutrophil differentiation marker Ly6G (Fig. 2c). While invasive L. monocytogenes infection (listeriosis) is not common, this systemic infectious disease has a much higher rate of hospitalization (up to 95%) and mortality (up to 30%) than those caused by most other foodborne pathogens [55–57]. Immunocompromised individuals, pregnant women, and the very young and elderly are particularly at risk for serious L. monocytogenes infections [55]. Several major outbreaks of listeriosis have also occurred from the consumption of contaminated food [58], indicating the significance of studying L. monocytogenes pathogenesis in a monomicrobial setting.

Fig. 2.

Fig. 2

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Comparison of i.v. and i.p. S. aureus infection. Wt mice on C57BL/6J were infected i.v. or i.p. with 5 × 107 cfu of S. aureus USA300, and their tissues were isolated at 24 h post-infection. A) Liver, lung, and spleen bacterial burden were determined by extracting live bacteria from tissues, plating serial dilutions onto TSA plates, and counting the number of colonies (n = 5). B) Liver sections (5 μm) were immunostained with human anti-PNAG antibodies and Alexa 647-conjugated rat anti-human monoclonal antibodies (original magnification, ×200). C) Liver sections were immunostained with Alexa 488-conjugated rat anti-mouse Ly6G monoclonal antibodies (original magnification, ×200)

The approaches described here are designed to determine the significance of HSPGs, identify critical HS modifications and structures, and characterize how HSPGs modulate pathogenesis and host defense in vivo. The role of HSPGs in systemic bacterial infection is probed by exogenous administration of purified heparan compounds, heparan derivatives, heparan antagonists, and degrading enzymes and genetically modified mice lacking HS biosynthetic enzymes or HSPG core proteins. General GAG biosynthesis inhibitors (e.g., xyloside, chlorate) have been used in studies in vitro, but these are not recommended for use in vivo because of their strong toxicity.

If HSPGs promote infection by serving as an attachment site for a particular bacterial pathogen, addition of purified HS or heparin will result in reduced tissue bacterial burden and other parameters of infection. On the other hand, if the HSPGs promote pathogenesis by inhibiting host defense, then administration of HS or heparin will enhance bacterial virulence by interfering with bacterial clearance. For example, delayed i.v. administration of heparin at 6 h post-infection significantly increases the liver and lung bacterial burden at 24 h post-infection in i.v. S. aureus infection (Fig. 3).

Fig. 3.

Fig. 3

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O-sulfated motifs in heparin promote i.v. S. aureus infection. Wt mice on BL/6J were infected i.v. with 5 × 107 cfu of S. aureus USA300, injected i.v. with 5 μg of porcine mucosal heparin (HP) or completely de-O-sulfated porcine heparin (deOS-HP) 6 h after infection, and the liver and lung bacterial burden were determined at 24 h post-infection (n = 5, *p < 0.05, ANOVA)

Heparin lyases and heparan derivatives are used to determine the essential structural features of HSPGs that modulate infection. Heparin lyases selectively digest certain domains of HS in HSPGs. For example, bacterial heparinase I and III digest sulfated and low sulfated regions of HS, respectively [59], thus allowing determination of whether sulfated or low sulfated HS domains are important in infection. Many chemically modified or size-defined heparin derivatives are also available, and these reagents are used to determine essential HS modifications and minimum active size of HS chains. For example, complete removal of O-sulfate motifs inhibits the ability of heparin to promote S. aureus i.v. infection, indicating that O-sulfate groups in HS mediate the ability of HSPGs to enhance S. aureus virulence (Fig. 3).

The response of Wt and KO mice lacking genes for certain HS modification enzymes or HSPG core proteins is compared to establish the physiological significance and relevance of a particular HS modification and HSPG in systemic bacterial infection. For example, Sdc1−/− mice significantly resist P. aeruginosa lung [30] and burned skin [26] infection compared to Wt mice, suggesting that Sdc1 promotes the pathogenesis of P. aeruginosa in these tissues. Similarly, Sdc1−/− corneas are significantly less susceptible to S. aureus infection compared to Wt corneas [25], suggesting that subversion of Sdc1 may be a broadly used bacterial virulence mechanism.

3.1. Intravenous Bacterial Infection

  1. Preparation of infectious inoculum. Grow 10 μl of S. aureus or L. monocytogenes from the glycerol stock in 5 ml TSB or BHI broth overnight at 37 °C with agitation. On the next day, dilute the overnight culture tenfold (e.g., 0.5 ml into 4.5 ml TSB or BHI broth) and regrow to mid- to late-log growth phase (OD600nm: ~0.6–0.8) (see Note 7). Estimate the bacterial concentration by turbidity (i.e., based on the predetermined growth curve). Wash a sufficient volume of the regrown bacterial culture for the experiment by centrifuging at 10,000 × g for 5 min, resuspending the bacterial pellet in PBS, and centrifuging at 10,000 × g for 5 min. Discard the supernatant and repeat the wash process once. Resuspend the bacterial pellet in PBS to the desired concentration. We generally inject 106–108 cfu of S. aureus and 104–106 cfu of L. monocytogenes for i.v. and i.p. bacteremia studies. Immediately plate out serial dilutions of the inoculum on TSA or BHI agar plates and count the number of colonies on the following day to determine the exact viable infectious inoculum.

  2. Place restraining device on a flat surface and at a comfortable height to insert the needle almost parallel to the tail vein.

  3. Prepare 1 ml syringe with bacterial inoculum. Remove any air bubbles from the syringe. We generally inject 0.1–0.2 ml of the infectious inoculum. Up to 1% of the body weight in volume can be administered per injection.

  4. Place the mouse in the restraining device (see Note 8). Locate one of the two lateral veins in the middle third of the tail. Turn mouse body 90° clockwise or counterclockwise if necessary. Wipe tail with an alcohol prep pad (see Note 9).

  5. Hold tail with thumb and index fingers of the non-dominant hand above the site of injection to apply digital pressure and occlude the vein. If a tourniquet is used to occlude the vein, restrain the tail by holding below the site of injection.

  6. Hold syringe with thumb, index, and middle fingers of the dominant hand closer to the bottom of the syringe. Holding closer to the bottom of the syringe helps to reach plunger quickly and push reagent into the vein.

  7. With the bevel of the needle facing up and almost parallel to the vein, insert the needle. The needle should slide in smoothly and a flash of blood in the hub of the needle will be seen (see Note 10).

  8. Release the manual occlusion and slowly push the syringe plunger to inject the infectious inoculum into the vein. If the needle is indeed in the vein, there will be no resistance while injecting and the vein will turn clear for a few seconds. If you feel resistance and see that the inoculum is leaking around the vein, remove the needle and retry above the first injection site.

  9. Remove the needle and apply gentle pressure with a sterile gauze pad until bleeding stops.

  10. Return mouse to the cage and observe to ensure that the bleeding has stopped.

  11. At various time points, euthanize mice by CO2 inhalation or anesthesia followed by cervical dislocation. Perfuse mice with 10 ml of PBS per mouse and carefully isolate the tissues (e.g., liver, lung, spleen) with fine scissors and forceps. If blood readouts are to be obtained, collect blood by cardiac puncture before whole body perfusion (see Note 11).

  12. Weigh tissues and place in a 100 mm petri dish with 3 ml of DMEM with 10% FBS (see Note 12). In the petri dish, strain lungs through a 70 μm filter using a plunger of a 5 ml syringe. Wash the strainer once with 1 ml DMEM with 10% FBS to remove loosely attached strained tissues.

  13. Transfer 1–2 ml of the strained tissue mixture to microcentrifuge tubes and centrifuge at 10,000 × g for 10 min. Discard supernatant and resuspend pellet in 500 μl TSB or BHI broth containing 0.1% Triton X-100.

  14. Incubate for 30 min at room temperature with vigorous vortexing every 10 min to lyse host cells and to recover both intracellular and extracellular bacteria. Viability of most bacteria, including S. aureus and L. monocytogenes, are not affected by 0.1% Triton X-100.

  15. Prepare serial dilutions of the detergent extract in TSB or BHI broth and plate onto TSA or BHI agar plates.

  16. Incubate overnight at 37 °C and count the colonies the following day. Based on the dilutions and weight of tissues, calculate the tissue bacterial burden per gram of tissue.

3.2. Intraperitoneal Bacterial Infection

  1. Prepare the bacterial inoculum as in step 1 of Subheading 3.1.

  2. Prepare a 1 ml syringe with the bacterial inoculum. Remove any air bubbles from the syringe. We generally inject 0.1 ml of the infectious inoculum per mouse for i.p. studies.

  3. Manually restrain the mouse with the abdomen facing you. Prepare the injection site with an alcohol pad.

  4. Slightly angle the needle (~20°) and insert ~5 mm through the abdominal wall of the animal’s left lower quadrant. Ensure internal organs, such as colon, bladder, and kidney, have not been penetrated by slight aspiration. Remove needle and discard.

  5. Follow steps 11–16 of Subheading 3.1 to quantify the tissue bacterial burden (see Note 11).

3.3. Cecal Ligation and Puncture-Induced Polymicrobial Sepsis

  1. Clean surgery area of lab bench with 70% ethanol. Warm up heating pad in microwave and place under the surgical metal board. Bring the sterilized tray to the bench and open the drape by touching only the outside to keep the sterile condition inside the drape. Open the lid of the tray. Place sterile 3 ml syringe with needle, 4-0 sutures with needle, and alcohol and gauze pads into the opened sterilization tray.

  2. Weigh mouse and anesthetize by injecting i.p. ketamine (100–120 mg/kg) and xylazine (5–10 mg/kg) (see Note 13). Pinch toe with a forceps to ensure that the mouse is anesthetized, which usually takes 5–10 min.

  3. Shave lower abdomen using a clipper. Shave well as excess hair is a cause of contaminating infection. Clean shaved area with an alcohol pad.

  4. Topically apply eye ointment to keep the eyes moist during the CLP procedure.

  5. Place mouse on the surgical metal board on its back, with its head positioned away from the operator. Secure legs on the surgical board with tape (autoclave indicator tape works well) (see Note 14).

  6. Disinfect the shaved abdomen with betadine solution and alcohol pads. Repeat three times.

  7. Change gloves. From here on, do not touch non-sterile materials. Change gloves when needed.

  8. Locate 2–2.5 cm below the bottom of the rib cage in the midline of the body. Gently lift skin with forceps. Make a small longitudinal incision (~1–2 cm) using fine dissecting scissors or a scalpel, being careful not to penetrate into the peritoneal cavity.

  9. After the initial incision, use sterile small scissors to slightly extend the incision and gain entry into the peritoneal cavity. Identify and dissect the linear alba of the abdominal musculature and make a small incision in the superficial fascia of the abdominal muscle, making sure not to nick underlying intestinal tissues. Locate the cecum, which should be just underneath the incision area. Place a piece of sterile gauze below the incision site.

  10. Gently exteriorize the cecum with blunt anatomical forceps and place it on the sterile gauze.

  11. Ligate cecum with 4-0 silk sutures at the desired location. Percent ligation is determined by the length from the ligated site to the distal cecum tip divided by the whole length of the cecum below the ileocecal valve (i.e., 100% ligation = ligation just below the ileocecal valve). The % ligation is one of parameters that determines the severity of sepsis (higher = more leakage of cecal flora, leading to more severe sepsis).

  12. Puncture through the ligated cecum 1–3 times with a 20G needle. The number of punctures is another parameter that determines sepsis severity. More punctures cause more leakage of cecal flora, which leads to more severe sepsis. Sepsis severity may also be controlled by the size of the puncturing needle.

  13. Gently extrude a small portion of fecal material to ensure patency of punctures (see Note 15). Carefully replace the cecum into the abdominal cavity.

  14. After replacing the cecum in the abdomen, close the fascia and abdominal musculature by applying simple running 4-0 silk sutures (2–3 stitches) and close the skin by metallic skin clips or running sutures (2–3 stitches). Clean the abdominal area with an alcohol pad.

  15. Resuscitate by injecting pre-warmed sterile saline (1.25 ml/25 g body weight, subcutaneously).

  16. Inject buprenorphine (0.05 mg/kg body weight, s.c.) to relieve pain from the surgery. Repeat every 12 h for 2 days after CLP.

  17. Place mouse in a clean cage on its back (see Note 16). Monitor every 12 h for 2 days after CLP.

  18. Follow steps 11–16 of Subheading 3.1 to quantify the tissue bacterial burden (see Note 11).

3.4. Heparan Compounds and Heparan Antagonists in Systemic Bacterial Infection (See Note 17)

  1. Dilute heparan compounds in PBS to the desired concentration. The dose range of heparan compounds to be tested should be chosen based on preliminary titration experiments.

  2. When co-administering heparan compounds with bacteria in the i.v. and i.p. infection models, resuspend washed bacteria in step 1 of Subheading 3.1 in the solution containing heparan compounds, co-infect, and follow subsequent steps for i.v. and i.p. bacterial infection as described in Subheadings 3.1 and 3.2. For CLP-induced sepsis, heparan compounds can be administered either i.v. or i.p. Follow subsequent steps for assessing the tissue bacterial burden CLP model as described in Subheading 3.3. Effects of heparan compounds on readouts other than tissue bacterial burden can also be examined with this approach (see Note 11).

    Heparan compounds can also be given before or after infection. Figure 3 shows that delayed i.v. injection of 5 μg of heparin at 6 h post-infection significantly increases the liver and lung bacterial burden at 24 h post-infection in i.v. S. aureus bacteremia.

  3. To assess the effects of heparan antagonists (see Note 17), such as protamine and surfen, dilute antagonists in PBS to the desired concentration and resuspend the washed bacteria in step 1 of Subheading 3.1 in the heparan antagonist solution for co-infection studies. The heparan antagonists can also be administered before or after i.v. or i.p. infection. Similarly, heparan antagonists can be injected i.v. or i.p. immediately after CLP, before CLP, or several hours after CLP in the CLP-induced sepsis model to study the effects of HSPG inhibition on the onset, progression, and outcome of sepsis. Follow subsequent steps to assess the tissue bacterial burden in i.v. (see Subheading 3.1) or i.p. (see Subheading 3.2) bacterial infection or CLP-induced sepsis (see Subheading 3.3) models. Other parameters of systemic infection can also be examined with this approach (see Note 11).

3.5. Heparin Lyases and Heparan Derivatives in Systemic Bacterial Infection (See Note 17)

  1. Dilute heparin lyases in PBS to the desired concentration. The effective dose range should be titrated in preliminary experiments. In studies examining the effects of heparinase II on intranasal P. aeruginosa infection, we found that 0.3 mU per mouse of heparinase II effectively removes HS from the surface of airway epithelial cells and attenuates bacterial virulence in newborn mice [30].

  2. Resuspend the washed bacteria in step 1 of Subheading 3.1 in the heparin lyase solution and follow subsequent steps to co-infect with bacteria in the i.v. (see Subheading 3.1) or i.p. (see Subheading 3.2) bacterial infection model or administer immediately after the CLP procedure in the CLP-induced sepsis model (see Subheading 3.3). The heparan lyases can also be injected before or after i.v. or i.p. infection or CLP. Follow subsequent steps in Subheadings 3.1, 3.2, and 3.3 to assess the tissue bacterial burden and other parameters of infection (see Note 11).

  3. To assess the effects of heparan derivatives (e.g., desulfated heparin compounds, oligosaccharides), dilute derivatives in PBS to the desired concentration and resuspend the washed bacteria as in step 1 of Subheading 3.1 and follow subsequent steps to co-infect with bacteria in the i.v. (see Subheading 3.1) or i.p. (see Subheading 3.2) bacterial infection model or co-administer in immediately after CLP in the CLP-induced sepsis model (see Subheading 3.3). Follow subsequent steps to assess the tissue bacterial burden in Subheading 3.1, 3.2 and 3.3. Effects of heparin derivatives on other readouts of systemic bacterial infection can also be examined with this approach (see Note 11).

    Heparan derivatives can also be injected before or after i.v. or i.p. infection or CLP. Figure 3 shows that delayed administration of heparin at 6 h post-i.v. S. aureus infection significantly increases the bacterial burden in the liver and lung, whereas completely de-O-sulfated heparin has no effect, indicating that O-sulfate motifs in heparin and HS enhance S. aureus virulence.

3.6. Genetically Modified Mice in Systemic Bacterial Infection

  1. Obtain genetically modified mice and appropriate Cre driver mice from the corresponding labs or from commercial sources.

  2. Infect i.v. or i.p. with S. aureus or L. monocytogenes or perform CLP on KO mice, corresponding Wt littermates, and conditional KO mice and their floxed and Cre driver controls identically and follow steps described in Subheading 3.1, 3.2, and 3.3 to assess the tissue bacterial burden (see Note 11 to assess other parameters of infection).

Acknowledgments

We would like to thank past and current members of the Park laboratory for developing the described procedures. This work was supported by NIH grants R01 HL132573 and R01 HL142213.

Footnotes

1.

All animal experiments must be approved by the Institutional Animal Care and Use Committee (IACUC) and comply with federal guidelines for research with experimental animals. All infectious agents administered to animals must be approved by the Institutional Biosafety Committee (IBC).

2.

C57BL/6 and BALB/c are the most frequently used inbred strains in mouse models of systemic bacterial infections. However, similar to sepsis in humans, several studies have reported that male mice are more susceptible to sepsis complications [60, 61]. Another note of caution is that BL/6 strains from JAX and Charles River have different origins, and they are genetically different and the designation “BL/6J” is used for Wt mice on the C57BL/6 background from JAX. Also, when comparing the response of Wt and KO mice, one should use Wt littermates obtained from heterozygous crosses of the KO line unless the KO line is congenic for a particular genetic background (i.e., backcrossed ≥10 times onto a particular background).

3.

It is important to confirm key data with at least two different bacterial strains to determine if the findings are bacterial strain-dependent.

4.

The tissue source of heparan compounds should be considered because HS from different tissues have a variable extent of modification (e.g., hepatic HS is more sulfated). Although we found that porcine heparin significantly promotes i.v. S. aureus bacteremia (Fig. 3) and P. aeruginosa pneumonia [30], they may not be active in other models of infection.

5.

Methods to chemically desulfate heparin are established and can be performed in-house [62–64]. Alternatively, HS from mutant CHO cells lacking certain modifications [65, 66] can be isolated and tested in the infection assays. Recombinant GAGs isolated from mutant CHO cells are also available commercially (Galen Laboratory).

6.

When using global or conditional KO mice to establish the significance of a particular HS modification or a particular HSPG, off-target effects of the mutation must be carefully considered. Ideally, to assess the direct causal role of HSPGs and their modifications in systemic bacterial infections, the KO mice should be phenotypically identical to their Wt littermates under unchallenged conditions. Inherent defects, especially in the immune system, can have profound effects on the readouts and outcome of systemic bacterial infections. Cre toxicity should also be considered, especially when ablating the gene of interest in hepatocytes using Albumin-Cre driver mice.

7.

Expression of bacterial virulence factors is regulated by the growth phase. In general, expression of cell surface virulence factors, such as adhesins, is high during early to mid-log growth, whereas expression of secreted factors such as exotoxins is high during late-log to stationary growth. The in vivo virulence of each bacterial species and strains at different growth phases should be determined in pilot studies.

8.

The restraining device should be cleaned frequently to prevent contamination.

9.

Mouse tails can be warmed to cause vasodilation by placing the mouse on a heating pad or by soaking the tail in warm water (~37 °C) for ~5 min.

10.

To ensure that the needle has been successfully inserted into the lateral vein, one can gently apply negative pressure to the syringe plunger after insertion of the needle to see a flash of blood returning to the syringe. However, because this practice can damage the veins, it should be avoided if planning to perform multiple injections within 24 h.

11.

The procedure described in Subheading 3.1, 3.2, and 3.3 measures the tissue bacterial burden, but this method can be easily adapted to assess other key parameters of systemic bacterial infection. Blood and serum prepared from blood can be used to perform complete blood count (CBC), serum chemistry, and cytokine analyses to assess the extent of the cytokine storm and multi-organ injury (MOI) and dysfunction (MOD). In addition to assessing the tissue bacterial load, isolated tissues can be homogenized and used for mRNA and protein measurements of cytokines, chemokines, and antimicrobial factors by RT-PCR or ELISA (kits available from various vendors: e.g., Biolegend, R&D Systems, Peprotech). Tissues can also be used for single-cell isolation and genomic, transcriptomic, proteomic, and metabolomics analyses at the single-cell level. Furthermore, isolated cells can be used to study direct interactions with the bacterial pathogen of interest.

Isolated tissues can also be processed for histopathological analyses. Paraffin-embedded or frozen tissue sections can be stained with hematoxylin and eosin to assess the extent of injury. Tissue sections can be stained with Gram’s solution or immunostained for poly-N-acetylglucosamine (PNAG), a cell surface polysaccharide expressed by many bacterial pathogens [53], to visualize bacteria, or immunostained with specific antibodies to examine the expression of HSPGs, inflammatory factors, and accumulation of leukocyte subsets (e.g., neutrophils, macrophages, lymphocytes). Figure 2b shows immunostaining of liver sections from mice infected i.v. or i.p. with S. aureus USA300 with anti-PNAG antibodies, and Fig. 2c shows immunostaining of infected liver sections with neutrophil-specific anti-Ly6G antibodies.

12.

Straining tissues in DMEM with 10% FBS prevents nonspecific adhesion of bacteria to plastic surfaces.

13.

An alternate anesthetic to ketamine/xylazine is isoflurane. Isoflurane anesthesia (2–4%) is given by inhalation, with scavenging either by house vacuum or fume hood. Recovery from isoflurane anesthesia is faster than ketamine/xylazine, thus it is important to rapidly perform the CLP procedure if using isoflurane as the anesthetic.

14.

Avoid securing mouse legs on the surgical board with pins and thumbtacks. This inflicts unnecessary pain to the animal.

15.

Extrude similar small amounts of fecal material for data consistency.

16.

Each CLP procedure for a mouse should not take more than 15–20 min to avoid additional anesthesia and potential complications from dehydration.

17.

Off-target and adverse effects of test compounds (e.g., heparan compounds and derivatives, antagonists, heparin lyases) on the bacteria and host should be determined. For adverse effects on the host, parameters such as weight loss, blood cell counts (by CBC analysis), and tissue injury (by histopathology or serum chemistry) should be assessed in mice administered with test compounds only, especially at the highest dose tested. The effects of test compounds on bacterial growth and viability should also be determined. For example, protamine has anti-bacterial activity at high concentrations in vitro, and it can also induce allergic inflammatory responses in vivo [67].

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