New Results

Staphylococcal Enterotoxin C promotes Staphylococcus aureus Infective Endocarditis Independent of Superantigen Activity

Kyle J. Kinney, Phuong M. Tran, Katherine N. Gibson-Corley, Ana N. Forsythe, Katarina Kulhankova, Wilmara Salgado-Pabón
doi: https://doi.org/10.1101/2019.12.13.875633

Abstract

The superantigen (SAg) staphylococcal enterotoxin C (SEC) is critical for Staphylococcus aureus infective endocarditis (SAIE) as tested in rabbits. Its hallmark function and most potent biological activity is hyperactivation of the adaptive immune system. Superantigenicity was proposed as a major underlying mechanism driving SAIE but was not directly tested. With the use of S. aureus MW2 expressing SEC toxoids, we show that superantigenicity does not contribute to vegetation growth, to the magnitude of myocardial inflammation or to acute kidney injury. In contrast, superantigenicity contributes to hepatocellular injury and overall systemic toxicity. Recent studies indicate that SAgs directly inhibit endothelial cell migration. We show that SEC inhibits production of serpin E1, crucial in cell migration and vascular repair. This may be central to SEC’s role in SAIE. This study highlights the critical contribution of an alternative function of SAgs to SAIE and broadens our current understanding of these molecules.

Introduction

Staphylococcus aureus infective endocarditis (SAIE) is an acute and invasive infection of the cardiac endothelium characterized by the appearance of vegetative lesions (1). S. aureus is the leading cause of infective endocarditis in the developed world (IE) (2-4). The pathognomonic vegetations are a meshwork of bacterial aggregates and host factors such as fibrin, fibrinogen, platelets, and red-blood cells that form predominantly on heart valves (5). SAIE results in significant damage to cardiac structures, in particular the valves and myocardium, due to tissue toxicity and abscess formation (6). Once established, SAIE can lead to severe complications, most notably congestive heart failure, stroke, acute kidney injury, and septic shock (4, 6, 7). Treatment of SAIE is challenging, requiring prolonged antibiotic therapy or surgery to remove infected valves (4, 6). Even with treatment, SAIE has a high rate of recurrence and a 22-66% mortality rate (2, 4). Infections are frequently associated with methicillin-resistant S. aureus (MRSA) which complicate treatment and increase mortality (8). Furthermore, life-saving medical interventions (i.e. valve replacement, cardiac devices, and hemodialysis), an increasing population with underlying conditions (i.e. diabetes mellitus and immunosuppression), and advanced age also increase the risk of acquiring S. aureus infections (2, 4). As a result, the incidence of SAIE in the developed world has continued to increase (4). Unfortunately, the great advances in cardiovascular medicine achieved in the last decade have failed to improve SAIE outcomes. Thus, the mechanistic understanding of the pathophysiology of SAIE is not only of fundamental interest, particularly as it relates to bacterial factors critical for vegetation formation and development of complications, but also of utmost importance for development of effective intervention strategies.

Epidemiological studies demonstrated a strong association between SAIE and a select group of superantigen (SAg) genes, where 18-25% of SAIE strains encode entC (staphylococcal enterotoxin C; SEC), 9-20% encode tstH (toxic shock syndrome toxin; TSST1), and 58-90% encode the enterotoxin gene cluster (egc) (9). Consistent with these studies, SEC, TSST1, and the egc SAgs SEI, SE like (l)-M, SEl-O, and SEl-U all contribute to IE and metastatic infection in experimental IE (10, 11). However, the underlying mechanism by which SAgs contribute to SAIE pathogenesis remains speculative. Classically, SAgs are known for their potent T cell mitogenic activity resulting in dysregulated activation and cytokine production leading to inflammatory syndromes, and, in extreme cases, toxic shock (12). Superantigenicity results from toxin cross-linking of the Vβ chain of the T-cell receptor (TCR) to the major histocompatibility complex class II (MHC-II) receptor on antigen presenting cells (12). Of relevance to SAIE, endothelial cells also express MHC-II, thus functioning as conditional antigen presenting cells capable of cross-linking Vβ−TCR resulting in endothelium-mediated superantigenicity (13).

The dysregulated immune activation caused by SAgs distracts and diverts the immune system (14). It also promotes multiple etiologies including atopic dermatitis, pneumonia, extreme pyrexia, purpura fulminans, and toxic shock syndrome (12). The commonly accepted model of the role of SAgs in SAIE includes localized or systemic superantigenicity that causes dysregulation of the immune system preventing clearing of S. aureus from the infected heart endothelium. SAgs also cause capillary leak and hypotension that alters the hemodynamics of the vascular system (12). This alteration of blood flow may enhance vegetation formation. However, the requirement of superantigenicity in the pathogenesis and pathophysiology of SAIE has not been directly tested. In this study, we addressed the hypothesis that superantigenicity promotes SAIE and disease sequelae.

We used the rabbit model of native valve IE with the well-characterized MRSA strain MW2 (SEC+) and MW2 stably expressing SEC toxoids (TCR or MHC-II/TCR inactivated) to provide evidence for the critical contribution of SEC but not superantigenicity to vegetation growth, to the magnitude of myocardial inflammation, and to injury to the renal and hepatic systems. We demonstrate that development of septic vegetations are a pre-requisite for embolic kidney injury and decreased renal function, while superantigenicity resulting from SAIE exacerbates embolic hepatocellular damage (even when exhibiting similar liver pathology) and exacerbates systemic toxicity. With the use of human aortic endothelial cells, we provide evidence that SEC selectively inhibits the pro-angiogenic factor serpin E1, demonstrating the ability of SEC to directly modify endothelial cell function in ways that can promote SAIE.

Results

Superantigenicity is not sufficient to promote vegetation formation

SAg activity is the most potent biological function of staphylococcal enterotoxins and causes lethal pathologies. To establish whether superantigenicity promotes development of SEC-mediated SAIE, we constructed a S. aureus strain expressing SEC with an inactive T-cell receptor (TCR) binding site (SECN23A). Asn23 is a highly-conserved, surface exposed residue located in a cleft between the O/B fold and β-grasp domain of SAgs (15). It forms hydrogen bonds with the backbone atoms of the complementarity-determining region (CDR) 2 of the Vβ-TCR (16). As such, Asn23 contact with the Vβ-TCR has one of the greatest energetic contributions of the complex. Mutations in this position, such as N23A or N23S, greatly destabilize the Vβ-TCR:SAg interaction with profound effects in SAg activity (16). SECN23A has no detectable binding to Vβ-TCR as measured by surface plasmon resonance, no proliferative T-cell responses in thymidine-incorporation assays at concentrations up to 30 μg/ml (16), no lethality or signs of TSS in rabbits vaccinated subcutaneously with 25 μg three times every two weeks (17), and no lethality in rabbits after intravenous injection at 3,000 μg/kg (18). Due to its biological inactivation, SECN23A is excluded as a select agent toxin (18). Importantly, several vaccination studies have shown no disruption in toxin structure and in the antigenic nature of the protein (17, 19-21).

MW2 (S. aureus SEC+), the isogenic deletion strain MW2Δsec (S. aureus SECKO), and MW2Δsec complemented to produce SEC with an inactive TCR-binding site (S. aureus SECN23A) were tested in the rabbit model of native valve IE and sepsis (10). Rabbits were inoculated intravenously with 2-4 ×107 total CFU after 2 hours of mechanical damage to the aortic valve and monitored for a period of 4 days. During that period, rabbits infected with S. aureus SECKO had a ∼66% decrease in overall vegetation formation where 8/12 rabbits had no vegetations (Fig. 1A and B) and 4/12 had very small vegetations (sent to pathology) with an estimated weight of 5 – 15 mg (Fig. S1). In stark contrast, S. aureus SEC+ formed vegetations in nearly all rabbits (14/15). Of the 14 hearts containing vegetations, 6 were sent to pathology. In the rest (8/9), vegetation size ranged from 11 – 116 mg with most vegetations weighing >25 mg (Fig. 1A and B). Surprisingly, complementation with SECN23A restored vegetation formation to wild type levels (13/17), with vegetation sizes ranging from 12 – 103 mg (Fig. 1A and B). Vegetations formed by S. aureus SEC+ and S. aureus SECN23A also had comparable bacterial counts of 1×107 – 4×109 CFU (Fig. 1C). Of importance, S. aureus SEC+ and S. aureus SECN23A exhibited similar levels of SEC production in liquid culture (Fig. S2) and similar growth rates and red blood cell hemolysis (Fig. S2). Yet on average, serum IL-6 concentration was reduced in rabbits infected with S. aureus SECN23A, consistent with decreased systemic inflammation (Fig. 1D). These results indicate that SEC has SAg-independent activity that is important for the establishment and progression of vegetative lesions.

Figure 1.
Figure 1. SEC is required for vegetation formation independent of superantigenic activity.

(A). Representative images of aortic vegetations (white arrow) (black arrow is a post mortem blood clot). (B) Total mean weights of vegetations dissected from aortic valves from rabbits infected with indicated strains S. aureus SECKO, SEC+, SECN23A, SECN23A/F44A/L45A. Error bars are represented by ± SEM. (C) Bacterial counts recovered from aortic vegetations from panel B. Bars represent median value. (D) Serum analyte levels of interleukin-6 (IL-6) from rabbits 48-96 hpi. Data are represented as mean (± SEM). The dashed line is the average analyte value of all rabbits pre-infection. (B-D) Statistical significance was determined by one-way ANOVA with the Holm-Sidak multiple comparison test with each SEC-producing strain compared to SECKO. (D) All groups were tested against pre-infection analyte values and were statistically significant, p < 0.0001. (B) **, p = 0.0037, ***, p = 0.0004. (C) **, p = 0.0023, ***, p = 0.0003. (D) *, p ≤ 0.0248. p values ≤ 0.05 are considered statistically significant.

SEC mechanism in SAIE is independent of MHC class II interactions

To confirm the SAg-independent contribution of SEC to SAIE, we constructed a S. aureus strain expressing SEC with dual inactivation of the TCR- and MHC class II-binding sites (SECN23A/F44A/L45A). MHC-II is also expressed by non-hematopoietic cells such as epithelial cells and endothelial cells (13). Hence, we also could not exclude the possibility that the in vivo SECN23A interactions with MHC-II accounts for the observed phenotypes. SEC Phe44 and Leu45, conserved among all enterotoxins, are located on a protruding hydrophobic loop that directly contacts MHC-II and forms strong electrostatic interactions with the α-chain (22, 23). Leu45 is the most extensively buried amino-acid residue in the SEC:MHC-II interface, but mutations in either residue (Phe44 or Leu45) effectively inactivate SEC binding (20-24). F44S alone is 1000-fold less efficient in MHC-II binding resulting in a concomitant reduction of IL-2 in T-cell proliferation assays (21, 25). Simultaneous introduction of N23A/F44A/L45A in SEC does not affect protein production, with the complemented strains showing no deficiencies in growth rates and hemolytic activity (Fig. S2).

S. aureus SECN23A/F44A/L45A was tested in the rabbit IE model as described above. As previously observed with S. aureus SECN23A, S. aureus SECN23A/F44A/L45A produced vegetations at wildtype levels (12/17), with vegetations that ranged in size from 3 – 107 mg, most weighed >25 mg (Fig. 1A and B) and contained 5×107 – 2×109 CFU (Fig. 1C). Four of the 12 hearts containing vegetations were sent to pathology. The S. aureus SECN23A/F44A/L45A strain on average also resulted in reduced serum IL-6 concentrations when compared to rabbits infected with S. aureus SEC+ (Fig. 1D). Overall, infection with S. aureus strains producing toxoids formed vegetations in 77% of the rabbits (26/34), compared to 93% (14/15) in rabbits infected with S. aureus SEC+. These results highlight the critical requirement of SEC in SAIE independent of superantigenicity and MHC class II interactions.

Superantigenicity does not drive myocardial inflammation in SAIE

SAIE presents as a rapidly-growing and progressive vegetative lesion that results in the quick destruction of valvular leaflets and extension of the infectious process into the myocardium and adjacent structures (5). So far, the data supports a critical contribution of SEC but not superantigenicity to vegetation growth on heart valves. We then asked whether SEC superantigenicity promotes extension of the vegetative lesion into the surrounding tissue changing the overall cardiac pathology. To address this, we performed histopathological analyses on transverse sections of hearts containing vegetations. Rabbits infected with S. aureus SECKO with no vegetations did not exhibit pathology at the end of experimentation (Fig. S1). Hence, we processed all hearts of S. aureus SECKO infected rabbits with visible vegetations (mean size 2.7 ± 1 mm2, n=4). Hearts from rabbits infected with S. aureus SEC+, S. aureus SECN23A, or S. aureus SECN23A/F44A/L45A were selected randomly on the basis of presence of vegetations (mean size 6.6± 2 mm2, n=15).

Consistent with histopathology of SAIE described in humans, S. aureus vegetations in rabbits were composed of large aggregates of bacterial colonies interspersed in a fibrinous meshwork of host factors and cell debris (Fig. S3-S5). However, the vegetative lesions were heterogeneous across infection groups in presentation (location and size of bacterial clusters) and in the magnitude of suppurative intracardial complications (myocardial inflammation and septic coronary arterial emboli). In rabbits infected with S. aureus SEC+(n=6), most vegetations were located on aortic valve cusps and valve leaflets, with large clusters of bacteria present on the leaflets and intermixed within the central core of the vegetation adjacent to the aorta. A few vegetations formed on the aortic wall were transmural (across the entire wall) and extended into the adjacent adipose tissue. Rabbits infected with S. aureus-producing SEC toxoids (SECN23A or SECN23A/F44A/L45A) exhibited very similar histologic presentation to each other and to those infected with S. aureus SEC+, with a few exceptions. The endothelium adjacent to the vegetation of S. aureus producing SEC toxoids was rarely hypertrophied (plump) and the vegetation was not observed to form on valve leaflets. Strikingly, all of the small S. aureus SECKO vegetations formed on the aortic wall and extended into the adjacent adipose tissue (Fig. S1).

To directly address the contribution of SEC superantigenicity to myocardial inflammation, the magnitude of the inflammatory cell infiltrate was graded on a scale of 0-3 histologically (Fig. 2A). Most vegetative lesions presented with inflammation that was almost exclusively heterophilic (neutrophilic) adjacent to the vegetations (Fig. 2A). Foci of heterophils infiltrating the myocardium, cellular debris, and necrosis were also observed (Fig. 2A, insets). In the most severe cases (Grade 3), large and coalescing bands of heterophilic infiltrate surrounded the aortic ring (Fig. 2A). Vegetative lesions from S. aureus SEC+ consistently showed high grade myocardial inflammation that were indistinguishable histologically from those formed by S. aureus producing SEC toxoids (SECN23A or SECN23A/F44A/L45A) (Fig. 2B). Surprisingly, the S. aureus SECKO vegetations that formed on the aortic wall, albeit small, caused high grade inflammation adjacent to the vegetation. This is in stark contrast to the histopathology from rabbits infected with S. aureus SECKO with no vegetations, which was unremarkable (Fig. S1). Of interest, septic coronary arterial emboli (coronary arteries containing fibrin or bacterial thrombi) with adjacent myocardial necrosis were observed in rabbits infected with S. aureus SEC+ and SECN23A (Fig. 2C and E). Vegetations that penetrated deeper into the pericardium causing epicardial lesions and saponification (necrosis) of epicardial fat were present in 5/15 rabbits infected with S. aureus SEC+ or S. aureus producing SEC toxoids (Fig. 2D and E). Coronary emboli and epicardial lesions were observed in only 1/4 rabbits infected with S. aureus SECKO (Fig. 2E). These observations indicate that intracardial complications, such as myocardial inflammation, arise as a result of the presence of a vegetation and are independent of SEC superantigenicity.

Figure 2.
Figure 2. Vegetative lesions present with bacterial infiltration, inflammation, and necrosis.

(A) Examples of histopathologic assessment of myocardial inflammation during infective endocarditis (Graded 0-3). Inflammation was graded based on the amount of inflammatory cell infiltrate noted within the myocardium. 0 = no inflammation, 1 = multifocal, scattered infiltrate (arrows), 2 = coalescing foci to bands of infiltrate, 3 = wide zones of diffuse infiltrate with necrosis. Bar = 100 µm, inset bar = 20 µm. (B) Scoring of myocardial inflammation from HE stained images 48-96 hpi. (C) Examples of a fibrinonecrotic focus (dotted outline; note the myocardial necrosis within the outline which is a lighter pink color), (*) a centrally located thrombus and intravascular bacteria (deeply basophilic/blue material). Left bar = 100 µm, right bar = 20 µm. (D) Examples of an epicardial lesion with saponification (necrosis) of epicardial fat (arrows) and a locally extensive zone of myocardial mineralization (encircled). Bar = 100 µm. (E) Histopathologic scoring the presence or absence of cardiac pathological findings: bacterial thrombi and associated necrosis, intravascular (IV) bacteria, and epicardial fibrin and inflammation.

SEC inhibits the pro-angiogenic factor serpin E1 in endothelial cells

In acute IE, vegetative lesions develop rapidly with no evidence of repair (26). The fact that SEC promotes SAIE independently of superantigenicity suggests that it can directly target the endothelium and modify its function. Re-endothelialization, driven by pro-angiogenic factors, is essential for vascular endothelial repair (27). We hypothesized that SEC may dysregulate angiogenesis as a mechanism to promote disease. To test this, immortalized human aortic endothelial cells (iHAEC) were treated with 20 μg/ml of purified SEC and a protein array utilized to measure changes in secreted angiogenesis-related proteins. Twenty-four soluble factors produced by iHAEC were consistently detected in supernates (6 anti-angiogenic, 15 pro-angiogenic, and 3 cytokines). A Log2 fold change of ± 1 was set as a threshold for relevant changes (Fig. 3). None of the anti-angiogenic factors exhibited relevant changes from baseline. Of the pro-angiogenic factors, serpin E1 [PAI-1 (plasminogen activator inhibitor-1)] exhibited on average an 81% decrease (Log2 = -2.38) from baseline. The cytokines IL-1β, IL-8, and MCP-1 (monocyte chemoattractant protein-1) were not detected in amounts above threshold (Fig. 3). The selective inhibition of the pro-angiogenic factor serpin E1 is consistent with the hypothesis that SEC inhibits angiogenesis in endothelial cells.

Figure 3.
Figure 3. SEC elicits anti-angiogenic responses in endothelial cells.

Relative protein abundance of secreted angiogenic factors from iHAECs treated with 20 µg/mL of SEC for 24hrs. Data are represented as mean (± SEM). Log2 fold change compared to a media control. Dashed line represents threshold for relevant changes set at ± 1.

SEC contribution to vegetation formation is sufficient to promote high lethality

Cardiotoxicity and septic shock are complications associated with SAIE that frequently lead to higher mortality rates in humans (4, 5, 28). We had hypothesized that one of the mechanisms leading to high lethality in S. aureus SEC+ IE is vascular toxicity and multi-organ dysfunction resulting from superantigenicity (10). Yet, infection with either S. aureus SECN23A or S. aureus SECN23A/F44A/L45A still led to high lethality, with ∼50% of rabbits succumbing to infection during the experimental period (8/17 for SECN23A and 10/17 for SECN23A/F44A/L45A) compared to 73% of rabbits infected with S. aureus SEC+ (Fig. 4A). Rabbits infected with SEC-producing strains (wildtype or toxoid) consistently exhibited higher bacteremia (>1×103 CFU/ml) than those infected with S. aureus SECKO (Fig. 4B), which correlates with the presence of large septic vegetations. All strains tested presented with similar degrees of splenomegaly due to infection compared to uninfected controls (Fig. 4C). Thus, superantigenicity alone does not fully account for the high lethal outcomes associated with SEC production. Instead, SEC contribution to vegetation formation plays a prominent role in SAIE lethality.

Figure 4.
Figure 4. Expression of SEC toxoids leads to systemic infection and lethality.

(A) Percent survival of rabbits infected intravenously with 2×107-4×107 CFU of indicated strain measured over 4 days. *, p = 0.0269, **, p = 0.0063, ***, p = 0.0007 log-rank Mantel-Cox Test. (B) Bacterial counts per milliliter of blood recovered from rabbits post mortem. Bars represent median value. (C) Enlargement of the spleen (splenomegaly) resulting from S. aureus infection. Data are represented as mean (± SEM). The dashed line is the average spleen size of uninfected control rabbits. (B-C) Statistical significance was determined by one-way ANOVA with the Holm-Sidak multiple comparison test with each SEC-producing strain compared to S. aureus SECKO. (B) *, p = 0.0339, **, p = 0.0028. p values ≤ 0.05 are considered statistically significant.

Vegetation fragmentation and metastatic infection occur in one third of SAIE episodes and are associated with hemodynamic and embolic complications in multiple organ systems, including the vascular, nervous, pulmonary, gastrointestinal, renal, and hepatic systems (4). Septic embolization of cardiac vegetations increases mortality in patients with IE (29). In our previous studies, we noticed that rabbits consistently developed lesions in the liver and kidneys when infected with S. aureus wild type strains in the IE model (30). Hence, to further tease out the contribution, if any, of superantigenicity to systemic complications associated with SAIE, we focused on the effect of SEC production to kidney and liver injury and function.

SEC causes renal impairment independent of superantigen-mediated toxicity

We had previously observed renal ischemia, infarction, and abscess formation associated with SEC production during SAIE in rabbits (10). It remained to be established if superantigenicity-mediated toxicity significantly contributed to acute kidney injury. To address this, all experimental rabbits were grossly assessed for kidney lesion pathology (n=61) on a scale from 0-3. The lesions presented as hemorrhagic, necrotic, or ischemic. In the most severe pathology (Grade 3), lesions were locally extensive, coalescing to diffuse, and extended across a large surface of the kidney (Fig. 5A, Table S3). Kidneys from S. aureus SEC+ infected rabbits presented with severe pathology (Grade 2-3) in 66% of the animals (Fig. 5B). Similar kidney pathology developed in 50% of rabbits infected with S. aureus producing SEC toxoids (8/17 for SECN23A and 9/17 for SECN23A/F44A/L45) (Fig. 5B). Overall, rabbits infected with SEC-producing strains (wildtype or toxoid) were more likely to develop severe kidney pathology compared to S. aureus SECKO infected rabbits (OR: 13.50, 95% CI: 1.931-150.2, p = 0.0037) (Fig. S6).

Figure 5.
Figure 5. SEC toxoids retain ability to cause metastatic infections and renal impairment.

(A) Kidney Gross Pathology Grading Scale (Grades 0-3). 0 = no lesions, 1 = rare, small (<4mm) multifocal lesions, 2 = numerous large (>5mm) multifocal lesions, 3 = extensive to coalescing to diffuse lesions. Blue arrows indicate ischemic lesions, red arrow indicates a hemorrhagic lesion. (B) Scoring of kidney lesions post mortem. Statistical significance was determined by the Fisher’s exact test comparing categorical pathology grades 0-1 with grades 2-3 between S. aureus SECKO with each SEC-producing strain. (C) Serum levels of analytes 48-96 hpi. Data are represented as mean (± SEM). The dashed line is the average analyte values of all rabbits pre-infection. Statistical significance was determined by one-way ANOVA with the Holm-Sidak multiple comparison test. All groups were tested against pre-infection analyte values and were statistically significant, p < 0.03. (B) *, p ≤ 0.0432, **, p = 0.0047. (C) *, p ≤ 0.0397, **, p = 0.0046, ****, p < 0.0001. p values ≤ 0.05 are considered statistically significant.

Consistent with kidney pathology, serum levels of blood urea nitrogen (BUN) and creatinine (biological markers of renal function) were significantly increased in rabbits infected with SEC-producing strains (Fig. 5C). BUN levels rose three-fold over pre-infection baseline in 89% of these rabbits (40/45) and creatinine rose two-fold in 44% (24/45). In stark contrast, few rabbits infected with S. aureus SECKO had dramatic fold changes over baseline, where only 17% (2/12) and 25% (3/12) exhibited similar increases in BUN and creatinine, respectively (Fig. 5C). These results provide evidence that acute kidney injury in experimental SAIE is likely due to embolic disease (vegetation fragmentation and lodging in the kidneys) leading to decreased renal function rather than kidney failure that is observed in toxic shock syndrome (12).

Superantigenicity promotes hepatocellular injury and systemic toxicity

SAIE can lead to acute liver injury through persistent systemic inflammation and hypoperfusion (31-33), effects that can be secondary to superantigenicity. In our studies, liver pathology presented as pale, streak-shaped lesions that where focal, multifocal, or coalescing (Fig. 6A). Lesions were grossly scored on a scale from 0-3 (Table S3). In the most severe pathology (Grade 3), lesions presented as multifocal to coalescing, and extensive to diffuse throughout the surface. Livers from S. aureus SEC+ infected rabbits presented with severe pathology (Grade 2-3) in 80% of the animals (12/15; Fig. 6B). Similar liver pathology developed in ∼70% of rabbits infected with S. aureus producing SEC toxoids (9/17 for SECN23A and 13/16 for SECN23A/F44A/L45) (Fig. 5B). Overall, rabbits infected with SEC-producing strains (wildtype or toxoid) were more likely to develop severe liver pathology compared to S. aureus SECKO infected rabbits (OR: 7.286, 95% CI: 1.806-26.91, p = 0.0064) (Fig. S6). Of note, in rabbits infected with S. aureus SECKO, severe liver pathology was largely observed only in rabbits that developed small aortic vegetations (3/4 rabbits). These results indicate that SEC production and vegetation formation are critical mediators of liver pathology, likely via the release of septic emboli based on gross lesions.

Figure 6.
Figure 6. Superantigenicity promotes hepatocellular damage.

(A) Liver gross pathology grading scale (grades 0-3). 0 = no lesions, 1 = rare, focal streak-shaped lesions, 2 = multifocal to coalescing streak-shaped lesions, 3 = multifocal streak-shaped extensive to diffuse lesions. White arrows point to streak-shaped ischemic lesions characteristic of grade 1-3, red arrows indicate wide-spread ischemic lesions characteristic of grade 3. (B) Scoring of liver pathology post mortem. Statistical significance was determined by the Fisher’s exact test comparing categorical pathology grades 0-1 with grades 2-3 between the S. aureus SECKO with each SEC-producing strain. (C) Serum levels of analytes 48-96 hpi. Data are represented as mean (± SEM). The dashed line is the average analyte or ratio value of all rabbits pre-infection. Statistical significance was determined by one-way ANOVA with the Holm-Sidak multiple comparison test with each SEC-producing strain compared to SECKO. All groups were tested against pre-infection analyte values and were statistically significant for AST and LDH with all SEC-producing strains significant for ALT, p < 0.005. Statistical significance for AST to ALT ratio was determined by the Fisher’s exact test comparing ratio values 0-1.8 to values > 1.8 between the strain SEC+ to superantigenic deficient groups (B) **, p ≤ 0.0071. (C) *, p ≤ 0.0151, **, p ≤ 0.0089. p values ≤ 0.05 are considered statistically significant.

To evaluate if superantigenicity, whether directly or indirectly, has an effect on liver function, we measured serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT; Fig. 6C). AST and ALT are biomarkers of hepatocellular damage. Interestingly, rabbits infected with S. aureus SEC+ had significantly higher mean levels of AST (236 U/L) and ALT (103 U/L) compared to those infected with S. aureus producing SEC toxoids or S. aureus SECKO (AST ≤ 73 U/L; ALT ≤ 76 U/L). Correspondingly, the ratio of AST to ALT was significantly higher in rabbits infected with S. aureus SEC+ (Fig. 6C). These rises in liver aminotransferase enzymes are consistent with what is observed in humans during acute liver injury and ischemic hepatitis (32-33). Of note, lactate dehydrogenase (LDH) was the only other enzyme, of those tested, differentially increased in rabbits infected with S. aureus SEC+ (Fig. 6C). LDH is found in almost every cell in the body, as such, is a non-specific biomarker of tissue damage. Therefore, superantigenicity increases overall tissue toxicity and hepatocellular injury, as reflected by increased levels of AST, ALT, and LDH.

Discussion

S. aureus SAgs are ubiquitous among human clinical isolates and implicated in both colonization and pathogenic mechanisms (34). Although their significance as virulence factors has been established, how SAgs specifically contribute to S. aureus pathogenesis has become a pressing question with the discovery of the multifunctionality of these toxins (35-37). Still, in SAg-mediated illnesses, superantigenicity is placed as a triggering event mediating or exacerbating pathological responses during S. aureus infection. Current evidence indicates that the SAgs SEC, TSST1 and select egc toxins play a novel and essential role in the etiology of SAIE by yet uncharacterized mechanisms (10, 11). It has been proposed that superantigenicity leading to hypotension and immune dysregulation allows bacterial immune evasion and persistence, while direct interaction with the heart endothelium promotes endothelium dysfunction and disease progression (10, 11, 35). With the use of S. aureus producing SEC inactivated in MHC-II and/or TCR binding, we provide evidence that superantigenicity is not the primary mechanism by which SEC promotes vegetation formation, cardiac toxicity, and extracardiac complications such as such acute kidney injury. Our results are consistent with published studies noting that not all SAgs, such as the egc SAgs SEG and SEl-N, promote SAIE (11). SEG and SEl-N have comparable T cell mitogenic activity as SEA, SEB, and TSST1 (38, 39), indicating that superantigenic defects are not likely to account for their deficiency in promoting SAIE. It further highlights the critical contribution of SAgs, like SEC, in life-threatening pathologies by novel mechanisms that remain largely speculative or poorly understood at best.

The fact that SAgs promote SAIE development irrespective of their superantigenic activity indicates that their ability to target the endothelium and modify its function, as demonstrated for TSST1, may be an important mechanism contributing to disease development (35). TSST1 directly causes dysregulated activation of iHAECs and disrupts vascular integrity and re-endothelialization (35). Re-endothelialization is essential for vascular repair and wound healing and is dependent on angiogenic signals (40). Here we show that SEC selectively inhibits the pro-angiogenic factor serpin E1. Serpin E1, also known as plasminogen activator inhibitor type-1 (PAI-1), is particularly induced as part of the wound-repair program where it promotes endothelial cell migration towards fibronectin (41). The specific inhibition of serpin E1 in human umbilical vein endothelial cells (HUVECs) disrupts angiogenesis in in vitro cell migration and capillary network formation assays (42). Thus, SEC could promote IE if the injured endothelium is delayed from healing, exposing the sub-endothelial tissues and promoting deposition of fibrin, other pro-coagulants and S. aureus, ultimately contributing to initiation or spread of the vegetative lesion (10, 34).

Once established, SAIE is locally destructive, extending beyond the valve leaflets or valve cusps into perivalvular tissue, including the aortic wall, causing progressive myocardial inflammation and abscess formation (5). Of great importance, the cardiac histopathology presentation of SAIE in rabbits is strikingly similar to that observed in humans. Myocardial inflammation is almost exclusively heterophilic causing aortic ring abscesses and necrosis in the most severe cases. Yet, myocardial inflammation resulting from S. aureus expressing SEC toxoids was not distinct from that caused by the wildtype strain. Therefore, while SEC is required for development of large septic vegetations, the data does not support a significant contribution of superantigenicity to the intracardiac heterophilic response that ensues following vegetation formation. It is unlikely that SEC solely drives this response as the atypical formation of small vegetations on the aortic wall in rabbits infected with S. aureus SECKO also leads to similar responses adjacent to the vegetation. S. aureus produces a multitude of secreted toxins and enzymes during invasive disease including the large family of cytolysins, proteases, and other SAgs that likely contribute to inflammation. Future studies will need to address the impact of any of these factors in SAIE cardiotoxicity.

SAIE has a high mortality rate owing to the high incidence of both intracardiac complications arising from the rapid local spread of the infection and the high incidence of embolization of septic vegetation fragments (2, 4, 43). Lodging of septic emboli within terminal blood vessels causes localized ischemia and infarction in multiple organ systems. In humans and in experimental rabbits, these complications can be manifested as myocardial infarction, kidney and/or liver injury, and strokes (43). Of these, kidney injury leading to acute renal insufficiency with progression to acute renal failure is tightly associated with SAIE severity, development of septic shock, and IE lethality (7). The role of SEC in kidney injury during SAIE has been demonstrated and it was proposed that SEC’s role in inflammation, toxicity, or immune dysregulation was also required (10). Our studies rule out superantigenicity-mediated immune dysregulation as the primary mechanism causing kidney injury. Rabbits infected with S. aureus producing SEC toxoids develop severe kidney pathology coupled with significant decreases in renal function. Furthermore, the type of lesions observed upon gross examination (hemorrhagic, necrotic, or ischemic) are consistent with embolization of cardiac vegetations. Hence, the contribution of SEC to kidney injury may be a consequence of its role in vegetation formation. However, we have not ruled out a mechanism of renal deterioration arising as a direct effect of SEC on the kidneys or its vasculature.

While the correlation between kidney function and poor prognosis in IE is well established, literature on the effects of SAIE on liver injury is scarce (4). The liver has central roles in clearing circulating bacteria and their toxins and is key in initiating or amplifying inflammatory responses during systemic infections (7). Development of liver emboli has been noted in patients with IE that develop septic shock, yet, not much more has been reported. In line with published reports, we found that multifocal ischemic liver lesions were present grossly in the great majority of rabbits infected with S. aureus SEC+. As seen with the kidney, liver pathology has a similar presentation in rabbits infected with strains producing wildtype SEC or toxoids. Again, these results demonstrate a dependency on SEC for tissue injury that is independent of superantigenicity. Hepatocellular injury as a result of extrahepatic bacterial infection has been reported to be largely dependent on bacterial toxins (7). Indeed, AST and ALT are increased >15 fold and >3 fold, respectively, in up to 40% of rabbits infected with S. aureus SEC+. In contrast to the kidneys, in the liver, superantigenicity significantly contributes to hepatocellular injury. Increases in serum aminotransferases correlates with increases in IL-6 and LDH in rabbits infected with S. aureus SEC+ versus those infected with S. aureus expressing SEC toxoids. Altogether, the data provides evidence for superantigenicity increasing hepatocellular injury during SAIE either directly or indirectly by increasing embolic events.

It is critical to recognize that while adaptive immune system activation is characteristic of staphylococcal SAgs, this is not their only biological function. Recently, the SAg SEl-X was found to inhibit neutrophil function via a sialic acid-binding motif uniquely present in this SAg (37). The SAgs TSST1, SEB, and SEC directly affect the function of endothelial/epithelial cells and adipocytes independent of superantigenicity (35, 44, 45). It was reported for TSST1 that activation of epithelial cells was caused by a dodecapeptide close to the base of the central α-helix of the molecule (36). The dodecapeptide sequence is found in all staphylococcal SAgs, yet its effects on non-hematopoietic cells is poorly characterized (12, 36, 45). The relevance of the SEC dodecapeptide in endothelial cell function and S. aureus diseases such as IE is currently being addressed.

In conclusion, we provide evidence that SEC is a multifunctional toxin critical to the pathogenesis and pathophysiology of SAIE. The superantigenicity independent effects of SEC are essential for the establishment of proliferative vegetations and systemic complications associated with disease progression. Overall, superantigenicity seems to exacerbate systemic inflammation and toxicity, with a significant contribution to hepatocellular injury. It now becomes possible to tease apart the localized SAg-host interactions triggering or exacerbating vegetation growth. It is also clear that SAgs do much more than previously anticipated or expected based on the current understanding of these molecules. Given the prevalence of SAgs among both methicillin-susceptible and resistant S. aureus strains, it becomes fundamental to understand the involvement of superantigenic-independent mechanisms in other invasive and life-threatening diseases.

Materials and methods

Bacterial strains and growth conditions

Staphylococcal strains were used from low-passage-number stocks. All staphylococcal strains were grown in Bacto™ Todd Hewitt (TH) (Becton Dickinson) broth at 37°C with aeration (225 rpm) unless otherwise noted. Strains and plasmids used in this study are listed in Table S1. Plasmids used for complementation were maintained using carbenicillin (100 µg/ml) in E. coli DH5α. For endocarditis experiments, strains were grown overnight, diluted, and washed in phosphate buffered saline (PBS - 2mM NaH2PO4, 5.7 mM Na2HPO4, 0.1 M NaCl, pH 7.4).

Construction of chromosomally complemented toxoid strains

SEC is a CDC designated select agent. As such, we are not allowed to use the wildtype copy of the gene in recombinant studies. For this reason, all PCR products generated in the making of the toxoid complement strains either included the permissible N23A SEC TCR mutation or was only a partial amplification of sec missing either the TCR or MHC-II domain. Each step of plasmid construction was verified by Sanger sequencing to contain the N23A TCR mutation. PCR amplification was performed using Phusion polymerase (New England Biolabs; NEB) unless otherwise noted. S. aureus expressing SECN23A or SECN23A/F44A/L45A were made by markerless chromosomal complementation in MW2Δsec (Table. S1) with the genes expressed under the control of the native promoter and terminator (46). The SECN23A gene sequence was created by amplifying two fragments from MW2 with primer sets pJB38xN23ApromF/promN23R and termN23F/pJB38xN23AtermR. The chromosomal complementation plasmid, pJB38-NWMN29-30, was digested with EcoRV and PCR products inserted by Gibson Assembly (NEB) as previously described, creating pKK29 (47). SECN23 was amplified from pKK29 with the primer set pUC19SECN23AptF/pUC19SECN23ApR and inserted into linearized pUC19, KpnI and EcoRI, by Gibson Assembly to create pKK33. The MHC-II binding site mutations were introduced into pKK33 by site-directed mutagenesis (QuickChange II, Agilent Technologies) using the primer set SECF44A/L45Afor/SECF44A/L45Arev, creating pKK39. The SECN23A/F44A/L45A gene sequence was amplified from pKK39 with primer set pJB38xN23ApromF/pJB38xN23AtermR and inserted into pJB38-NWMN29-30 as described above, creating pKK42. pKK29 and pKK42 were electroporated into S. aureus RN4220 and moved into MW2Δsec by generalized transduction with ϕ11(48). S. aureus strains containing plasmid were selected for with chloramphenicol (20 µg/ml) at 30°C. Allelic exchange was performed as previously described (46), chromosomal insertions detected by PCR with primer set XNWMN2930F/XNWMN2930R, and verified by Sanger sequencing. Primers were purchased from Integrated DNA Technologies (Table S2).

Rabbit model of IE

The rabbit model of IE was performed as previously described with some modifications (10). 2-3 kg New Zealand White Rabbits were obtained from Bakkom Rabbitry (Red Wing, MN) and anesthetized with ketamine (dose range: 10-50 mg/kg) and xylazine (dose range: 2.5-10 mg/kg). Mechanical damage to the aortic valve was done by introducing a hard, plastic catheter via the left carotid artery, left to pulse against the valve for 2h, removed, and the incision closed. Rabbits were inoculated via the marginal ear vein with 2×107-4×107 total CFU in PBS and monitored 4 times daily for a period of 4 days. For pain management, rabbits received buprenorphine (dose range: 0.01 – 0.05 mg/kg) twice daily. At the conclusion of each experiment, bacterial counts were obtained from heparinized blood (50 USP units/mL). Rabbits were euthanized with Euthasol (Virbac) and necropsies performed to assess overall health. Spleens were weighed and used as an infection control, kidney and liver gross pathology was graded using gross lesion pathology scale (Table S3), aortic valves were exposed to assess vegetation growth, and vegetations that formed were excised, weighed, and suspended in PBS for CFU counts. A minimum of 4 rabbit hearts from each infection group were placed in 10% neutral buffered formalin and further processed by the Comparative Pathology Laboratory at the University of Iowa for histopathological analyses. Vegetation weight and bacterial counts cannot be obtained from hearts prepared for histology. All experiments were performed according to established guidelines and the protocol approved by the University of Iowa Institutional Animal Care and Use Committee (Protocol 6121907). All rabbit experimental data is a result of at least 3 independent experiments per infection group.

Histopathologic scoring

Fixed tissues were routinely processed, cut at 5 µm, and hematoxylin and eosin (HE) stained or Gram stained. Slides were reviewed and scored by a board-certified veterinary pathologist.

Serum analysis

Rabbit serum was obtained from heparinized blood (50 USP units/mL) collected before infection and at 48, 72, and 96 hpi. Blood was centrifuged at room temperature at 5000 x g for 10 min. The collected supernatant was centrifuged for an additional 5 min, filter sterilized using a 0.2 μm filter, and stored at -80°C for further analysis. Serum samples were sent to the University of Iowa Diagnostic Laboratories and evaluated for the following serum analytes: aspartate aminotransferase (AST; U/L), alanine aminotransferase (ALT; U/L), blood urea nitrogen (BUN; mg/dl), creatinine (mg/dl), and lactate dehydrogenase (LDH; mg/dl).

Rabbit IL-6 ELISA

IL-6 was quantified from serum samples using the R&D Systems DuoSet Rabbit IL-6 ELISA kit, according to manufacturer’s instructions. Serum samples were diluted 1:10 in reagent diluent prior to use. The optical density (O.D.) was determined using a TECAN M200 plate reader (Tecan Group Ltd.) set to 450 nm with wavelength correction set to 540 nm. A standard curve was created by linear regression analysis of the IL-6 concentration versus OD, log-transformed (GraphPad Prism 8).

SEC purification

SEC was purified from S. aureus strain FRI913 in its native form by ethanol precipitation and thin-layer isoelectric focusing as previously described (49). Preparation of SEC resulted in a single band by Coomassie blue stain. Toxin preparations were tested for lipopolysaccharide (LPS) contamination with the ToxinSensor Chromogenic LAL Endotoxin Assay following manufacturer’s instructions (GenScript). SEC preparations had < 0.1ng of LPS per 100 µg of toxin (< 0.02 ng of LPS per 20 µg of SEC).

Human aortic endothelial cell culture

Immortalized human aortic endothelial cells (iHAECs) were cultured as previously described in Medium 200 with low-serum growth supplement (both from Gibco Life Technologies) in 5% CO2 at 37°C (35). All experiments were conducted using iHAECs at 4-10 passages from a single clone.

Proteome Profiler™ Human Angiogenesis Antibody array

96-well tissue culture plates coated with 1% gelatin were seeded with 7,000 iHAECs/well and grown to confluence. Fresh media containing purified SEC (20 µg/ml) was added and plates were incubated overnight at 37°C with 5% CO2. The supernatant was removed and stored at -80°C for further analysis. The relative expression of 55 angiogenesis-related proteins was determined from the supernatant using a Proteome Profiler™ Human Angiogenesis Antibody Array according to the manufacturer’s instructions (R&D Systems). 120 µL of supernates along with IRDye 800CW Streptavidin (LI-COR, 1:2000 dilution) as a secondary antibody were used for this assay. The fluorescent signal was detected using the LI-COR Odyssey CLx (84 µm resolution, auto intensity 800 nm channel). Mean pixel density was calculated from duplicate spots on the membrane and averaged using Image Studio Software (LI-COR). The log2 fold-changes over media-only control were calculated for each detected protein. All treatments were matched to media only control. Data is a result of four biological replicas performed in duplicate.

Statistical analyses

The log-rank, Mantel Cox test was used for statistical significance of survival curves. Normality was assessed using the D’Agostino & Pearson test along with associated Q-Q plots for data distribution. For comparison across means log-transformed data was used and statistical significance was determined by using one-way analysis of variance (ANOVA) with the Holm-Sidak multiple comparison test for the following data sets: vegetation size, vegetation CFU, blood CFU, spleen size, BUN, creatinine, AST, ALT, LDH, and IL-6. Statistical significance for gross pathology data was determined using Fisher’s exact test along with calculated odds ratios and 95% confidence intervals. Statistical significance of virulence factor production was determined by using the nonparametric Kruskal-Wallis test. p ≤ 0.05 was considered statistically significant (GraphPad Prism 8).

Funding

This work was supported by National Institutes of Health (NIH) grant R01AI34692-01 to W.S-P, NIH grant 5T32AI007511-23 to P.M.T., and NIH grant T32GM008365 to K.J.K.

Author Contributions

K.J.K. and W.S-P conceptualized and designed experiments, analyzed the data, and wrote the manuscript. K.J.K, P.M.T, A.N.F, K.K, and W.S-P carried out in vivo rabbit experiments. K.N.G-C provided intellectual and technical support on histopathological analysis and gross pathological grading. P.M.T and A.N.F carried out in vitro experiments for the proteomics array and serum analysis. All authors reviewed the manuscript.

Competing Interests

The authors declare no other competing interests.

Data and materials availability

All the data needed to evaluate the conclusions in this manuscript are present in the manuscript and/or supplementary materials.

Acknowledgements

We thank the University of Iowa Comparative Pathology Laboratory and University of Iowa Diagnostic Laboratories for histology and serum analysis services.

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Posted December 13, 2019.
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Staphylococcal Enterotoxin C promotes Staphylococcus aureus Infective Endocarditis Independent of Superantigen Activity
Kyle J. Kinney, Phuong M. Tran, Katherine N. Gibson-Corley, Ana N. Forsythe, Katarina Kulhankova, Wilmara Salgado-Pabón
bioRxiv 2019.12.13.875633; doi: https://doi.org/10.1101/2019.12.13.875633
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