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. Author manuscript; available in PMC: 2021 Dec 1.
Published in final edited form as: J Thromb Haemost. 2020 Sep 20;18(12):3236–3248. doi: 10.1111/jth.15065

Yersinia pestis escapes entrapment in thrombi by targeting platelet function

Samantha G Palace 1, Olga Vitseva 2, Megan K Proulx 1, Jane E Freedman 2, Jon D Goguen 1, Milka Koupenova 2
PMCID: PMC8040536  NIHMSID: NIHMS1681405  PMID: 33470041

Abstract

Background:

Platelets are classically recognized for their role in hemostasis and thrombosis. Recent work has demonstrated that platelets can also execute a variety of immune functions. The dual prothrombotic and immunological roles of platelets suggest that they may pose a barrier to the replication or dissemination of extracellular bacteria. However, some bloodborne pathogens, such as the plague bacterium Yersinia pestis, routinely achieve high vascular titers that are necessary for pathogen transmission.

Objectives:

It is not currently known how or if pathogens circumvent platelet barriers to bacterial dissemination and replication. We sought to determine whether extracellular bloodborne bacterial pathogens actively interfere with platelet function, using Y pestis as a model system.

Methods:

The interactions and morphological changes of human platelets with various genetically modified Y pestis strains were examined using aggregation assays, immunofluorescence, and scanning electron microscopy.

Results:

Yersinia pestis directly destabilized platelet thrombi, preventing bacterial entrapment in fibrin/platelet clots. This activity was dependent on two well-characterized bacterial virulence factors: the Y pestis plasminogen activator Pla, which stimulates host-mediated fibrinolysis, and the bacterial type III secretion system (T3SS), which delivers bacterial proteins into the cytoplasm of targeted host cells to reduce or prevent effective immunological responses. Platelets intoxicated by the Y pestis T3SS were unable to respond to prothrombotic stimuli, and T3SS expression decreased the formation of neutrophil extracellular traps in platelet thrombi.

Conclusions:

These findings are the first demonstration of a bacterial pathogen using its T3SS and an endogenous protease to manipulate platelet function and to escape entrapment in platelet thrombi.

Keywords: blood platelets, extracellular traps, plague, thrombosis, Yersinia pestis

1 ∣. INTRODUCTION

Platelets are anucleate cell fragments constituting the major blood component responsible for thrombosis. Activation of platelets and the clotting cascade leads to thrombus formation that prevents interstitial bleeding during vessel damage. A thrombus, in addition to platelets, contains a mesh of crosslinked fibrin monomers that enhance clot stability.1,2 Platelet activation and clot formation are important in hemostasis, but can also lead to thrombotic vessel occlusion.

Recently, platelets have been identified as key contributors to the innate and adaptive immune responses.3 These roles are reflected in the deep evolutionary entanglement of the vertebrate hemostatic and immune systems.4-6 The crosstalk between these two systems is thought to play an important functional role in effective pathogen control. For example, platelets express pathogen-associated molecular pattern receptors,7-12 including Toll-like receptors (TLRs), that directly activate and strengthen innate immune responses against both viral10,13 and bacterial infections.10,14-16 Signaling through these receptors allows platelets to stimulate the formation of neutrophil extracellular traps (NETs),8,13,17,18 which aid in the capture and removal of bacteria from the circulation but can also increase thrombosis.17,19 Furthermore, bacteria such as Escherichia coli,20 Streptococcus pyogenes,21 and Streptococcus sanguis22 induce platelet aggregation, resulting in the entrapment of bacteria in platelet thrombi. Controlled thrombosis in the context of infection can thus result in platelet activation, fibrin deposition, and NETosis, which together may work to confine pathogens and prevent dissemination while recruiting canonical immune cells.

Because platelets are highly abundant in the blood, their immune functions presumably pose a substantial barrier to the proliferation of bloodborne pathogens. However, it is not known how such pathogens avoid entrapment in platelet thrombi or clearance via platelet-mediated immune cell recruitment and activation. Yersinia pestis, the gram-negative etiological agent of plague, is a classic model system for bloodborne bacterial pathogenesis. Yersinia pestis is transmitted primarily via flea bite: after an infected flea deposits <1000 bacteria into the dermis of a susceptible mammalian host, Y pestis must establish dense, fulminant bacteremia to ensure successful colonization of new fleas feeding upon the infected mammal.23,24 The ability of Y pestis to reach the bloodstream and proliferate to high titer in the vasculature is thus an indispensable step in its transmission cycle.

Yersinia pestis encodes virulence factors that support bacterial proliferation and invasiveness by suppressing the mammalian innate immune system. One of the most important of these virulence factors is a type III secretion system (T3SS), which allows Y pestis to deliver certain bacterial proteins, called effectors, into the cytoplasm of targeted host cells. T3SS effector delivery (or “intoxication” of target cells) is crucial for Y pestis to evade innate immune responses, as it allows the bacterium to subvert leukocyte function.12,25 However, the effect of this system on platelet function is not known.

In addition to T3SS-mediated intoxication of innate immune cells, there is substantial evidence that Y pestis alters hemostasis and thrombosis as part of its transmission strategy. One of the most-studied virulence factors of Y pestis is Pla, a bacterial outer membrane protease that activates mammalian plasminogen, which in turn degrades fibrin.26,27 In the absence of Pla-mediated fibrin degradation during infection, resulting either from Pla-deficient Y pestis or from plasminogen-deficient mice, Y pestis is no longer able to efficiently establish bacteremia. Instead, fibrin and inflammatory cells accumulate around foci of bacterial growth, preventing dissemination28 and dramatically reducing virulence.29-31 These observations imply that altering thrombus formation may be crucial for the bacterial dissemination that drives high mortality in Y pestis infections. This model is consistent with the observation that heterozygous factor V Leiden mice infected with Y pestis32 have reduced mortality.

As platelets promote both thrombosis and local inflammation—defensive responses that can cripple the ability of Y pestis to produce systemic infection—we hypothesized that this pathogen targets platelet prothrombotic function to achieve successful dissemination. Here, we examine the interactions of Y pestis with freshly isolated human platelets in an in vitro thrombosis model. We show that Y pestis interferes with platelet thrombosis, and that this activity is dependent on both the bacterial T3SS and the plasminogen activator Pla.

2 ∣. RESULTS

2.1 ∣. Y pestis decreases platelet activation and aggregation

To assess whether Y pestis stimulates platelet aggregation, we incubated washed human platelets with the Y pestis strains JG150L or JG152L. JG150L is an attenuated Y pestis strain that carries a deletion of the pgm locus required for iron acquisition in vivo (Table 1), which allows for experimentation under biosafety-level 2 conditions; JG152L is a derivative of this strain that lacks the T3SS, and is discussed in detail in the next section. Platelets were mixed with or without Y pestis in HEPES buffered Tyrode solution supplemented with Ca2+/Mg2+ and fibrinogen, and aggregation was measured in a platelet aggregometer (PAP-8). Although previous work has reported platelet aggregation in response to other types of bacteria,21,22,33 JG150L did not lead to any observable platelet aggregation under these conditions, although platelets in this assay were able to aggregate robustly in response to stimulation with the thrombin receptor activating peptide (Figure 1A,B).

TABLE 1.

Bacterial strains and relevant characteristics

Strain Strain No. Genotype Relevant Characteristics Source
JG150L JG598 Δpgm/pMT1+ pCD1+ pPCP1+ pML001+ Functional T3SS and Pla 36
JG152L JG597 Δpgm/pMT1+ pCD1− pPCP1+ pML001+ Functional Pla, lacks T3SS apparatus and effectors 36
JG150L plaD86A JG908 Δpgm plaD86A/pMT1+ pCD1+ pPCP1+ pML001+ Functional T3SS, catalytically inactive Pla This study
JG152L plaD86A JG910 Δpgm plaD86A/pMT1+ pCD1− pPCP1+ pML001+ Catalytically inactive Pla, lacks T3SS and effectors This study
JG150LΔT3SE JG715 Δpgm/pMT1+ pCD1 (yopHΔ3-497 yopEΔ40-197 yopKΔ4-181 yopMΔ3-408 ypkAΔ3-731 yopJΔ-288 yopTΔ3-320) pPCP1+ pML001+ Functional Pla, functional T3SS apparatus, in-frame deletions of all seven T3SS effector genes 36
JG150LΔT3SE::+yopH JG680 Δpgm/pMT1+ pCD1 (yopEΔ40-197 yopKΔ4-181 yopMΔ3-408 ypkAΔ3-731 yopJΔ4-288 yopTΔ3-320) pPCP1+ pML001+ Functional Pla, functional T3SS apparatus, functional YopH effector, in-frame deletions of all other T3SS effector genes 36
JG150LΔT3SE::+yopE JG681 Δpgm/pMT1+ pCD1 (yopHΔ3-467 yopKΔ4-181 yopMΔ3-408 ypkAΔ3-731 yopJΔ4-288 yopTΔ3-320) pPCP1+ pML001+ Functional Pla, functional T3SS apparatus, functional YopE effector, in-frame deletions of all other T3SS effector genes 36
JG150LΔT3SE::+yopK JG682 Δpgm/pMT1+ pCD1 (yopHΔ3-467 yopEΔ40-197 yopMΔ3-408 ypkAΔ3-731 yopJΔ4-288 yopTΔ3-320) pPCP1+ pML001+ Functional Pla, functional T3SS apparatus, functional YopK effector, in-frame deletions of all other T3SS effector genes 36
JG150LΔT3SE::+yopM JG683 Δpgm/pMT1+ pCD1 (yopHΔ3-467 yopEΔ40-197 yopKΔ4-181 ypkAΔ3-731 yopJΔ4-288 yopTΔ3-320) pPCP1+ pML001+ Functional Pla, functional T3SS apparatus, functional YopM effector, in-frame deletions of all other T3SS effector genes 36
JG150LΔT3SE::+ypkA JG684 Δpgm/pMT1+ pCD1 (yopHΔ3-467 yopEΔ40-197 yopKΔ4-181 yopMΔ3-408 yopJΔ4-288 yopTΔ3-320) pPCP1+ pML001+ Functional Pla, functional T3SS apparatus, functional YpkA effector, in-frame deletions of all other T3SS effector genes 36
JG150LΔT3SE::+yopT JG685 Δpgm/pMT1+ pCD1 (yopHΔ3-467 yopEΔ40-197 yopKΔ4-181 yopMΔ3-408 ypkAΔ3-731 yopJΔ4-288) pPCP1+ pML001+ Functional Pla, functional T3SS apparatus, functional YopT effector, in-frame deletions of all other T3SS effector genes 36
JG150LΔT3SE::+yopJ JG686 Δpgm/pMT1+ pCD1 (yopHΔ3-467 yopEΔ40-197 yopKΔ4-181 yopMΔ3-408 ypkAΔ3-731 yopTΔ3-320) pPCP1+ pML001+ Functional Pla, functional T3SS apparatus, functional YopJ effector, in-frame deletions of all other T3SS effector genes 36
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FIGURE 1.

FIGURE 1

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Platelet aggregation is inefficient in the presence of Y pestis. Platelets isolated from healthy human blood were suspended in HEPES-buffered Tyrode's buffer and incubated with fibrinogen and the wild-type Y pestis strain JG150L or a Y pestis strain lacking the type III secretion system, JG152L, at MOI = 1. Aggregation was measured using a PAP-8 aggregometer at 37°C with constant stirring at 1000 rpm. A, Representative of platelet aggregation curves in the presence of Y pestis or TRAP (thrombin receptor activating peptide), a positive control for aggregation. B, Quantitation of aggregation measured as in panel A from n = 3 (2 F; 1 M) different human donors. C, Representative aggregation curves of platelets in the presence of Y pestis JG150L and thrombin (IIa, 0.1 U/mL). (D, E) Quantitation of (D) disaggregation and (E) area under the curve measured as in panel C from n = 4 (2 F, 2 M) different donors. Significance was calculated using one-way ANOVA, followed by Bonferroni correction; *P < .05

Thrombin (factor IIa) is the major physiological stimulus for platelet aggregation. When platelets were stimulated with thrombin in the aggregation assay, we observed robust, stable thrombus formation in the absence of Y pestis. This is contrary to stimulation of human platelets with thrombin receptor activating peptide, which had variable aggregation ability for different donors (Figure S1). In the presence of JG150L, however, thrombin-induced aggregation was both incomplete and unstable, and platelets disaggregated completely approximately 10 minutes after initial aggregate formation (Figure 1C-E). These observations suggest that Y pestis possesses one or more mechanisms that specifically target the ability of platelets to form stable aggregates.

2.2 ∣. The Y pestis protease Pla mediates bacterial destabilization of platelet thrombi

In normal mammalian hemostasis, the host zymogen plasminogen is converted to the active serine protease plasmin, which degrades crosslinked fibrin in clots leading to thrombus dissolution.1,34 The Y pestis protease Pla proteolytically activates mammalian plasminogen to plasmin, resulting in increased fibrin degradation and enhancing dissemination of Y pestis from peripheral infection sites.26,27 To determine whether plasminogen activation by Pla allows Y pestis to escape entrapment in platelet/fibrinogen clots, we incorporated human plasminogen into the in vitro platelet aggregation assay with thrombin stimulation as described in the previous section. The Y pestis strain JG150L, which carries a functional pla allele, destabilized the initial platelet thrombus and ultimately led to complete platelet disaggregation (Figure 2A,C). In contrast, a bacterial strain expressing a catalytically inactive Pla (JG150L plaD86A) was completely unable to destabilize the thrombus in this assay (Figure 2A,C). A similar pattern of Y pestis-mediated platelet disaggregation was observed in both the presence and absence of purified plasminogen (Figure 2A, curve #2 compared with Figure 1C, curve #2); this is likely because contaminating plasminogen in the purified fibrinogen added to the assay buffer and endogenous plasminogen released from platelet α-granules upon thrombin stimulation35 contribute to fibrinolysis. Taken together, these observations suggest that Y pestis destabilization of platelet thrombi requires proteolytic activation of plasminogen by Pla.

FIGURE 2.

FIGURE 2

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Y pestis destabilization of platelet thrombi in vitro requires Pla and the type III secretion system. Platelets from healthy human blood were suspended in HEPES-buffered Tyrode's buffer. Platelets were stimulated with thrombin (0.1 U/mL) in the presence various Y pestis strains (MOI = 1), fibrinogen, and plasminogen. Platelet thrombus formation was measured using a PAP-8 aggregometer. A, Representative aggregation curves of platelets in the presence of Y pestis strains expressing the T3SS and either the active protease Pla (JG150L, wild-type) or catalytically inactive Pla (JG150L plaD86A). Similarly, B, representative aggregation curves of platelets in the presence of a Y pestis strain that lacks the type III secretion system (T3SS) but expresses active Pla (JG152L), or a strain that lacks the T3SS and expresses catalytically inactive Pla (JG152L plaD86A). In all cases, “control” indicates platelets without bacteria or IIa stimulation. C, Quantitation of aggregation as in panels A and B from n = 4 (2 F, 2 M) different human donors. Shaded circles indicate individual donors. Significance was calculated using one-way ANOVA, followed by Bonferroni correction. D, Localization of Y pestis JG150L (+T3SS) and JG152L (−T3SS) during thrombin-stimulated platelet aggregation under the same conditions as in panels A and B, visualized with a Cascade 1K (Photometrics) EM-CCD camera using a 30 s exposure window (with constant stirring of the sample). Bioluminescence signal (light areas) corresponds to live Y pestis; the opaque stir bar is visible as a dark smear at the bottom of the assay tube in the 0 min panel. Representative images of three different platelet donors are shown

2.3 ∣. The Y pestis type III secretion system undermines platelet function and enables bacterial escape from platelet thrombi

The Y pestis T3SS delivers bacterial effector proteins into the cytosol of targeted host cells via a needle-like injectisome apparatus.12 To evaluate the effect of the Y pestis T3SS on thrombus formation, we performed platelet aggregation experiments using the Y pestis strains JG150L, which contains a functional T3SS, and JG152L, which lacks the pCDl plasmid that encodes the T3SS. Like JG150L, JG152L did not induce platelet aggregation in the absence of thrombin stimulation (Figure 1A,B). Surprisingly, however, T3SS-deficient strains were no longer able to interfere with the formation of platelet thrombi, even if they expressed functional Pla (Figure 2B,C).

To determine whether Y pestis becomes trapped in platelet thrombi, and whether the T3SS allows bacterial escape, we monitored the localization of Y pestis throughout the aggregation assay. JG150L and JG152L are both bioluminescent, allowing us to track the spatial distribution of bacteria in real time by directly imaging the distribution of bioluminescence in the assay tube (see Methods). The bioluminescence signal from the T3SS+ strain JG150L initially concentrated in the platelet thrombus following the addition of thrombin (platelets and bacteria aggregated around the stir bar, visible at the bottom of the assay tube), but bacteria escaped into solution within 30 minutes (dispersed luminescence throughout the tube, visible in Figure 2D and Movie S1), consistent with the platelet disaggregation we observe in the presence of this strain (eg, Figure 2A). Unlike JG150L, the T3SS-deficient strain JG152L remained entrapped in the platelet thrombus for the duration of the experiment (Figure 2D and Movie S2), despite its functional Pla allele and the presence of purified human plasminogen in the assay. Furthermore, when we excluded platelets from this assay, Y pestis was only partially able to disaggregate the resulting fibrinogen aggregate (Figure S2), indicating that fibrinolysis resulting from the presence of Pla and plasminogen does not fully explain Y pestis-mediated thrombus dissolution. These observations demonstrate that the T3SS is necessary for Y pestis-mediated platelet disaggregation and bacterial escape from platelet thrombi, and that the presence of functional Pla is necessary but not sufficient for thrombus destabilization.

2.4 ∣. T3SS effector proteins are dispensable for preventing platelet aggregation but required for disaggregation of activated platelets

The T3SS of Y pestis delivers seven bacterial effector proteins into target cells.12 These effector proteins exert a wide array of activities, including derangement of signaling pathways, interfering with remodeling of the actin cytoskeleton, and disruption of focal adhesion complexes leading to inhibition of phagocytosis.12 Because the effector proteins have distinct biochemical activities and target different aspects of host cell biology, we sought to identify the effector(s) responsible for subverting platelet thrombotic function.

We first tested the ability of selected Y pestis mutants to prevent platelet aggregation when bacteria and platelets were coincubated before thrombin stimulation. When platelets and the T3SS+ strain JG150L were incubated together for 10 minutes before thrombin activation, platelet aggregation in response to thrombin stimulation was completely inhibited (Figure 3A). As expected, the T3SS− strain JG152L, which lacks both the injectisome apparatus and the effector proteins of the T3SS, did not prevent platelet aggregation, showing that the T3SS apparatus as a whole is required for inhibiting platelet aggregation in response to thrombin. However, JG150LΔT3SE, which expresses a functional injectosome apparatus but lacks all seven of the effector proteins, inhibited thrombus formation as effectively as the T3SS+ JG150L strain in this assay (Figure 3A). When platelets are exposed to Y pestis before prothrombotic stimulation, therefore, none of the effector proteins are required to prevent platelet aggregation in response to thrombin: the injectisome apparatus alone is sufficient.

FIGURE 3.

FIGURE 3

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T3SS effector proteins are dispensable for preventing thrombus formation, but not for dissolution of established thrombi. A, Platelets from healthy human blood were suspended in HEPES-buffered Tyrode's buffer and coincubated with plasminogen, fibrinogen, and Y pestis at MOI = 1 for 10 min before stimulation with 0.1 U/mL of thrombin for an additional 20 min. Aggregation was measured using a PAP-8 aggregometer. Final aggregation (20 min after thrombin stimulation) is shown for platelets coincubated with the T3SS+ strain JG150L (+T3SS), the T3SS-deficient strain JG152L (−T3SS), or the strain JG150LΔT3SE (ΔT3SE), which expresses the T3SS injectisome apparatus but lacks all seven effector proteins. n = 5 (4F, 1M) different human donors. B, Platelets from healthy human blood were suspended in HEPES-buffered Tyrode's buffer in the presence of fibrinogen and plasminogen and stimulated immediately with thrombin. Y pestis was added at MOI = 1 after observable thrombus formation began. Aggregation was measured using a PAP-8 aggregometer. Final aggregation of platelets from n = 3 (3F) different donors was measured following the addition of the Y pestis strains JG150L (+T3SS), JG152L (−T3SS), the JG150LΔT3SE strain (ΔT3SE) that expresses the T3SS apparatus but lacks all effectors, and strains that express the injectisome apparatus with a single effector (JG150LΔT3SE::yopH, or +yopH; and JG150LΔT3SE::yopE, or +yopE). Note that reduced aggregation in this assay requires active reversal of thrombus formation, rather than the prevention of platelet activation shown in panel A. Significance was measured using one-way ANOVA, followed by Bonferroni correction

We reasoned that T3SS-mediated disaggregation of activated platelets when bacteria were added after thrombin stimulation (Figure 2D, top) might be a more complex process, requiring additional components of the T3SS. To evaluate bacterial escape from platelet thrombi, we initiated standard clot formation by incubating washed platelets with thrombin in the presence of fibrinogen and plasminogen, waited for clot formation to begin, and then added Y pestis strains at the time that thrombi began to form (~4 minutes after thrombin addition). This approach allowed us to define the bacterial factors required for thrombus disaggregation, as opposed to preventing initial thrombus formation. As in the previous experiments, JG150L (T3SS+) reversed platelet aggregation, whereas JG152L (T3SS−) did not, indicating that T3SS was required for thrombus disaggregation under these conditions (Figure 3B). In this assay, the JG150LΔT3SE strain failed to initiate thrombus disaggregation (Figure 3B, third column from left), indicating that the injectisome apparatus is not sufficient for thrombus dissolution in the absence of effector proteins.

To determine which of the effectors is responsible for platelet thrombus disaggregation, we used a panel of seven Y pestis strains that each express the injectisome apparatus and only one of the effector proteins.36 These strains enabled us to measure the ability of each individual effector protein to reverse platelet aggregation. We found that strains expressing either the tyrosine phosphatase YopH or the GTPase activating protein YopE were able to fully reverse platelet aggregation (Figure 3B). Each of the remaining effector proteins was only able to partially rescue the JG150LΔT3SE phenotype (Figure S3).

2.5 ∣. The Y pestis T3SS increases bacterial survival and reduces release of neutrophil DNA in platelet thrombi

Neutrophils have diverse antimicrobial functions including phagocytosis, degranulation to release proinflammatory and bactericidal compounds, and NETosis, during which neutrophil DNA is released into the extracellular environment to capture and neutralize bacterial pathogens. In vivo, platelets contribute to NET formation by either initiating or accelerating NETosis.8,10,13,19 Additionally, NETs in the circulation are highly prothrombotic.37 To understand the effect of neutrophils on bacterial viability and survival during thrombus formation, we adapted the in vitro platelet aggregation assay to include primary human neutrophils. Using a physiological proportion of 40 to 50 platelets to one neutrophil,10,13 we observed that the bioluminescence of the T3SS-deficient strain JG152L decreased significantly in a thrombus containing both platelets and neutrophils (Figure 4A-D and Figure S4A), indicating reduced survival of the T3SS− strain in these conditions, as maintaining bioluminescence from this system requires metabolic activity. Although aggregation results in some initial decrease of luminescence, perhaps because of increased opacity of the aggregate, the presence of neutrophils in the aggregate results in a further decline in luminescence of the T3SS- strain JG152L after aggregation is complete (Figure 4C,D and Figure S4D,E). Bacterial killing in this assay appears to be primarily attributable to neutrophil function, as aggregating platelets alone were not sufficient to reduce bioluminescence of JG152L (Figure 4B, Figure S4B,C). By contrast, the presence of the T3SS in the strain JG150L appears to be protective against bacterial killing in this assay (Figure 4A-D and Figure S4A).

FIGURE 4.

FIGURE 4

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The Y pestis T3SS increases bacterial survival and decreases NET formation in thrombi containing platelets and neutrophils. Y pestis with the T3SS (JG150L) or without the T3SS (JG152L) was incubated in siliconized glass tubes with thrombin, fibrinogen, plasminogen, and platelets and/or neutrophils (as specified) isolated from the same human donor. Bacterial bioluminescence was imaged as a proxy for bacterial viability. Luminescence was quantified for each frame as described (see Methods) and normalized to the total luminescence signal at time 0 (defined as 100%). Reduced bioluminescence is indicative of reduced survival. Survival curves of Y pestis in the presence of (A) fibrinogen only; or (B-D) fibrinogen and (B) platelets, (C) neutrophils, (D) platelets and neutrophils (40-50:1). Graphs show the mean and SEM of n = 3 (3F) different donors, for JG150L (solid line, orange shading) and for JG152L (dashed line, gray shading). (E-F). Fluorescent microscopy of thrombi formed during incubation of Y pestis with platelets and neutrophils isolated from the same donor. Neutrophil DNA released in the thrombus after 13 min of incubation in the presence of (E) Y pestis with T3SS (JG150L), or (F) Y pestis without T3SS (JG152L). Images were captured with a Nikon A1 confocal microscope at 100×, representative of n = 3. Antibodies used are as follows: α-CD41a FITC (platelets), red: α-CD66b APC (neutrophils), blue: DAPI (DNA)

To determine if neutrophils release their DNA during neutrophil-mediated killing of Y pestis in platelet/polymorphonuclear neutrophil (PMN) aggregates, we examined thrombi by fluorescent microscopy. We observed robust aggregation of neutrophil DNA, platelets, and bacteria in the platelet thrombi in the presence of the T3SS-deficient strain JG152L (Figure 4F). By contrast, there was a reduced level of neutrophil DNA release in the thrombus that formed around the wild type (T3SS+) strain JG150L (sparse DAPI staining, Figure 4E).

2.6 ∣. The Y pestis T3SS limits morphological changes in activated platelets

Activated platelets undergo profound morphological changes, including the formation of extensive lamellipodia that ultimately develop into spaghetti-like fibers that support tight thrombus formation.38 Because the T3SS effector proteins YopH and YopE that interfere with thrombus stability are known to affect the cytoskeleton of target host cells,39 we hypothesized that platelets intoxicated by the Y pestis T3SS may fail to undergo the appropriate morphological changes following thrombin stimulation. To assess the effect of the Y pestis T3SS on platelet morphological changes, we examined thrombi containing fibrinogen, platelets, plasminogen, and Y pestis by scanning electron microscopy (SEM) after 15 minutes of thrombin stimulation (0.1 U/mL) (Figure 5A-F). The thrombi formed by platelets incubated with JG150L were smaller and had looser morphology with identifiable individual platelets (Figure 5A,C,D), whereas thrombi formed in the presence of the T3SS-deficient strain JG152L were larger, more uniform, and contained no distinguishable individual platelets (Figure 5B,E,F). We found that the Y pestis T3SS limits the formation of extensively branched lamellipodia on platelets (Figure 5G-M, Figure S5). Interestingly, platelets found free from the thrombus following incubation with JG150L have an elongated, but not highly branched, morphology (Figure 5J,K, Figure S5A). This is distinct from the rounded morphology of resting platelets when they are not exposed to thrombin or to Y pestis (Figure 5G). When aggregation was performed in the presence of the T3SS-deficient JG152L strain, platelets underwent dramatic morphological changes (Figure 5L,M, Figure S5B) similar to those reported for thrombin-stimulated platelets in the absence of bacteria10 (Figure 5H,I). The T3SS-dependent ability of Y pestis to destabilize platelet thrombi may therefore act via effector-mediated inhibition of prothrombotic platelet cytoskeleton remodeling.

FIGURE 5.

FIGURE 5

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Morphological changes of platelets and platelet thrombi in the presence of Y pestis. Platelets were incubated 1:1 with Y pestis with the T3SS (JG150L) or Y pestis without the T3SS (JG152L) in the presence of thrombin (0.1 U/mL), fibrinogen, and plasminogen for 13 min in a PAP-8 aggregometer. Morphological changes of platelets were visualized by scanning electron microscopy (SEM). (A-F). Different levels of magnification of thrombi formed by (A, C, D) Y pestis with the T3SS (JG150L), or (B, E, F) Y pestis without the T3SS (JG152L). (G,M) Morphology of platelets from healthy donors incubated with (G) control (no treatment), (H, I) thrombin, (J, K) Y pestis with the T3SS (JG150L), and (L,M) Y pestis without the T3SS (JG152L). Arrows point toward individual Y pestis bacilli

3 ∣. DISCUSSION

In addition to their role in hemostasis, platelets perform various antimicrobial and signaling functions in the context of innate immune responses. However, it is not known how bloodborne bacterial pathogens overcome platelet thrombotic functions in the vasculature. Here, we show that the bacteremia-causing pathogen Y pestis uses specific virulence factors and strategies to undermine platelet function that might otherwise result in entrapment of the bacterium in thrombi. Most gram-negative bacteria20,40 can stimulate platelets directly, likely via interaction of lipopolysaccharide (LPS) from the bacterial outer membrane with the pattern-recognition receptor TLR4.8 Although Y pestis is a Gram-negative bacterium in the same family as E coli, we found that incubation of Y pestis with platelets does not result in platelet aggregation. Previous studies have demonstrated that, at 37°C and during mammalian infection, Y pestis produces an unusual tetra-acylated LPS that inhibits rather than activates TLR4 signaling and reduces innate immune responses to Y pestis infection33; this is in contrast with the canonically hexa-acylated, TLR4-stimulatory LPS produced by most other Gram-negative pathogens, as well as by Y pestis growing at 20° to 25°C and during infection of the flea vector. This alteration of LPS structure as a function of temperature illustrates the effectiveness of Y pestis innate immune evasion as a strategy to enhance dissemination. Although avoidance of TLR4 activation by Y pestis is thought to be particularly important in the context of leukocyte responses, it is possible that this strategy also enhances virulence by avoiding platelet TLR4 activation. This is a subject for further study.

Furthermore, we found that Y pestis interferes with thrombin-mediated platelet activation. In an in vitro thrombosis model, Y pestis destabilizes clot formation of freshly isolated human platelets and reduces neutrophil-DNA release in platelet/neutrophil thrombi. This derangement of platelet function requires both bacterial plasminogen activation (by Pla) and the Y pestis T3SS. The Y pestis protease Pla activates host plasminogen in vivo, promoting host fibrinolysis and inhibiting efficient thrombosis during infection.26 Pla is also known to enhance invasiveness of Y pestis and to support the bacteremia caused by this pathogen. The Pla-mediated release of Y pestis from destabilized platelet thrombi is a novel observation, supporting the model that Pla promotes virulence in part by preventing Y pestis from being trapped in thrombi.

Additionally, we found that the Y pestis T3SS alters platelet morphology, leads to clot disaggregation, and reduces neutrophil-DNA release in thrombi containing both platelets and neutrophils. The involvement of the T3SS in perturbing platelet function suggests that current models of T3SS function during infection should be expanded. The T3SS of Y pestis is known to be particularly important in undermining phagocytosis and other antimicrobial functions of macrophages and neutrophils,25,39,41-43 but the complete range of target cell types is not known. Target cell types are difficult to predict, because engagement of host cells by the Y pestis T3SS does not require a host protein binding partner. Instead, the apparatus secretes two translocon proteins, YopB and YopD, which are thought to interact with cell membrane lipids to form a pore to which the injection needle attaches.44 This is the first example of any bacterial T3SS targeting platelets, though it seems likely that the absence of previous reports simply reflects the relative rarity of bacteria/platelet interaction studies.

In our in vitro assays, the Y pestis T3SS is able not only able to reduce thrombus formation, but also to dissociate thrombi once formed. Although the dissolution of large, established thrombi by Y pestis is unlikely to take place in vivo, it is a dramatic and intriguing feat of host cell manipulation by the T3SS. YopH and YopE, the effectors that are each capable of mediating thrombus dissolution, have both been reported to interfere with actin cytoskeleton remodeling.39 These effectors may affect platelet function by inhibiting degranulation, altering cell morphology changes associated with activation, or both. Multiple host protein interacting partners have been identified for both the tyrosine phosphatase YopH (FAK, p130Cas, paxillin, Fyb, SKAP-HOM, PRAM-1, SLP-76, Vav, PLCγ2, p85, Gab1, Gab2, Lck, and LAT) and the GTPase-activating protein YopE (RhoA, Cdc42, Rac1, Rac2, and RhoG).12,38 Platelets express the transcripts for almost all of these proteins.45,46 Formation of platelet lamellipodia and filopodia requires Rac138 and Cdc42,47,48 whereas cell morphology and actin cytoskeleton remodeling is dependent on FAK49 and PI3K-p8550 pathways. Determining which of these proteins mediates the T3SS effect on platelet function and granule release will require careful genetic modeling, and is a critical future direction for understanding the molecular basis of the thrombus destabilization we report here. Although this work focused only on strong agonist stimulation, to recapitulate both the aggregation and the coagulation of a disseminated Y pestis infection, the use of alternative platelet agonists may be a tool to further understand the platelet activation pathways that are targeted by the Y pestis T3SS.

Our results showing the role of the Y pestis T3SS in bacterial survival in thrombi containing platelets and neutrophils are concordant with previous reports demonstrating the importance of YopH and YopE for Y pestis survival in coculture with primary human PMNs.36,42 Because these same effector proteins interfere with platelet function, we have not yet developed an experimental system to distinguish between the effects of YopH and YopE on neutrophils as opposed to their effects on platelet functions. Direct interactions between the T3SS and neutrophils likely play an important role in reducing bacterial killing and neutrophil-DNA release in thrombi: in particular, YopE is known to inhibit Rac2-mediated ROS production in neutrophils,51 a key mediator of NETosis.52 However, the result that YopH and YopE interfere with platelet thrombotic function, together with the established role for platelets in activating neutrophil functions and NETosis,8 warrants further investigation.

When platelets were exposed to bacteria before thrombin stimulation, YopH and YopE were not needed to interfere with platelet function: the effectorless Y pestis strain JG150LΔT3SE was able to prevent platelet aggregation. Although this strain lacks cytoplasmic effector proteins, it expresses a functional injectisome apparatus, including the translocon proteins YopB and YopD that form a 2-3 nm pore in the target cell membrane.53 YopB/D pore formation likely plays a role in the platelet inhibition we observe from this strain. Consistent with this model, the Y pestis injectisome and translocon proteins have been shown to cause cell death in macrophages.54

Platelets have direct antimicrobial effects, and also enhance or activate antimicrobial functions of other immune cell types, including neutrophils.55 We do not yet know the extent to which platelet activation contributes to effective immune responses in the context of Y pestis infection, though the appearance of NETs in thrombosis is suggestive, as are previous reports that link thrombosis to enhanced innate immune responses to Y pestis.27,56 The ability of Y pestis to undermine platelet activation lends credibility to the hypothesis that platelets represent an important barrier to the proliferation and transmission of bacteria in the vascular compartment. Future work will focus on exploring the contribution of platelets to immunity in in vivo infection models of Y pestis. A bubonic plague murine infection model will allow us to examine the hypothesis that platelets may pose a particularly important barrier to bacteria invading from the periphery—the physiological route of infection for Y pestis and many other pathogens. Platelet-mediated entrapment of Y pestis in aggregates during the early stages of infection might effectively prevent systemic infection in a manner that would not be observed in simplistic infection models such as intravenous infection.

The prevalence of type III secretion systems in Gram-negative pathogens suggests that T3SS-dependent subversion of platelet responses may be a conserved strategy among bloodborne Gram-negative pathogens, especially in light of the apparent dispensability of T3SS effector proteins. Independently of their relevance to disease processes, analysis of the interaction of bacterial pathogens with mammalian cells has produced important contributions to basic cell biology. Our observation that Y pestis manipulates platelet functions suggests that extending this strategy to encompass platelets will be a useful tool for probing the internal platelet machinery. This study is the first to report that platelets are targeted by the T3SS of a bloodborne bacterial pathogen, and provides support for the hypothesis that pathogens of the vasculature have evolved active strategies to circumvent platelet-mediated barriers to bloodstream infection.

4 ∣. MATERIALS AND METHODS

4.1 ∣. Bacterial strains and growth conditions

Yersinia pestis strains are presented in Table 1. Yersinia pestis strains were cultured in defined Serum Nutritional Medium57 supplemented with 2.5 mmol/L CaCl2 to suppress type III secretion during growth. All Y pestis strains in this study carry the pML001 plasmid encoding the bioluminescence operon lux from Photorhabdus luminescens,58,59 and so media were supplemented with 100 μg/mL ampicillin to maintain this plasmid.

The Pla-deficient strain JG150 plaD86A was constructed by introducing the pla gene with a D86A substitution that renders the Pla protein catalytically inactive60 into JG150 via allelic exchange with the suicide vector pRE107 as described.61 The bioluminescent derivative JG150L plaD86A was constructed by introducing the pML001 plasmid into this strain. The T3SS-deficient derivative of this strain was constructed by curing the pCD1 plasmid, encoding the T3SS, from JG150 plaD86A. Briefly, spontaneous pCD1 segregants were selected by supplementing tryptose broth agar with sodium oxalate to create a calcium-deplete growth environment as described,62 the loss of the pCD1 plasmid was confirmed by PCR, and the pML001 plasmid was introduced to the resulting strain to yield JG152L plaD86A.

4.2 ∣. Platelet and neutrophil isolation

Whole blood was collected from healthy adult human volunteers in compliance with protocols reviewed and approved by the University of Massachusetts Medical School Institutional Review Board. Washed platelets (and platelet-free neutrophils, where needed) were isolated as described.10

4.3 ∣. Platelet aggregation

Platelets were diluted to a final concentration of 2 × 108/mL in HEPES buffer (140 mmol/L NaCl, 6.1 mmol/L KCl, 2.4 mmol/L MgSO4-7H20, 1.7 mmol/L Na2HPO4, 5.8 mmol/L sodium HEPES, 0.35% BSA, and 0.1% dextrose) in 225 μL. Aggregation was performed in the presence of 3 μmol/L purified human fibrinogen, 1 mmol/L CaCl2, and 2 mmol/L MgCl2. Where specified, luminescent Y pestis (2 × 108/mL), neutrophils (5 × 106/mL), and/or 2 μmol/L purified human plasminogen (Haematologic Technologies, Essex Junction, VT; cat# HCPG-0130) were also included in the reaction. Final ratio of bacteria to platelets to PMNs was 40:40:1. Aggregation was induced with the addition of 0.1 U/mL purified human thrombin (factor IIa, Enzyme Research Laboratories, IN; cat. #HIIa), added 1 minute after components were mixed together unless otherwise specified. Platelet aggregation was monitored stirring at 37°C in a PAP-8 aggregometer as described.10

4.4 ∣. Monitoring entrapment of Y pestis in thrombi by bioluminescence

Localization of luminescent Y pestis was monitored in a custom-built apparatus. Briefly, Y pestis strains were mixed with platelets and/or neutrophils under the same experimental conditions used for platelet aggregation experiments described previously. Assays were performed in siliconized glass PAP-8 aggregometer tubes, incubated at 37°C with constant stirring, as in the PAP-8 machine. Luminescence signal was captured with a Cascade 1K (Photometries) EM-CCD camera using a 30-second exposure window. Luminescence intensity was analyzed with ImageJ. Intensity was measured in each frame for three regions: an “upper” region (a rectangle located in the upper half of the tube), a “lower” region (a polygon capturing the area immediately surrounding the stir bar), and the stir bar region itself. Signal intensity of the stir bar region was multiplied by two as an approximate correction for the opacity of the stir bar, and this intensity was then added to that measured for the other two regions of interest. The “total” luminescence metric in each frame was normalized to the luminescence measured in the first frame of the experiment to calculate change in luminescence over time. Luminescence of Y pestis from the Photorhabdus luminescens lux operon correlates with bacterial viability.36,63

4.5 ∣. Fluorescent microscopy of platelet thrombi

Thrombi formed as described previously containing Y pestis strains, platelets, fibrinogen, and neutrophils were fixed 13 minutes after the addition of thrombin. For confocal microscopy, samples were fixed with IC Fixative buffer (Ebioscience cat. #00-8222-49) added 1:1, according to manufacturer's instructions, and then stained with α-CD41a FITC (platelet marker) (Ebioscience cat. #11-0419-42), α-CD66b APC (neutrophil marker) (Ebioscience cat. #17-0666-42), and DAPI (DNA), and imaged on a spinning disk confocal Nikon TE2000E2 inverted microscope. Images were collected on a Photometries Coolsnap HQ2 camera.

4.6 ∣. Scanning electron microscopy

SEM was performed as described.10 Briefly, isolated platelets, at concentration of 4 × 105 cells in 200 μL were incubated with Y pestis as described in the figure 5 legend. Cells were fixed with a 2.5% glutaraldehyde in Sorenson phosphate buffer (0.1 mol/L, pH 7.4) for 2 hours. After fixation, platelets were washed three times in phosphate buffered saline (pH 7.4) for 5 minutes, and then fixed with 1% Osmium tetroxide in 0.1 mol/L phosphate buffered saline (pH 7.4) for 1 hour. The samples were visualized with a Quanta 200 FEG MKII SEM (FEI Company).

Supplementary Material

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Essentials.

  • Platelets entrap bacterial pathogens and mediate immune responses to bacteria.

  • The bacteremic pathogen Yersinia pestis must circumvent platelet barriers to dissemination.

  • Y pestis subverts platelet function using its type III secretion system and the Pla protease.

  • These Y pestis virulence factors decrease NETosis, thrombus stability, and enhance bacterial survival.

ACKNOWLEDGMENTS

UMMS Electron Microscopy Core is supported by NIH Instrument grant S10RR021043 and NIH Ultramicrotomy award SI0OD021580. Support for this project: NIAID T32 AI095213 to S. G. Palace; NIH grants N01-HC 25195, U01HL126495, UH3TR000921-04 to J. E. Freedman; and AHA grant 16SDG30450001 and NIH grant R01 HL153235 to M. Koupenova.

Funding information

National Institutes of Health, Grant/Award Number: N01-HC 25195, U01HL126495 and UH3TR000921-04; American Heart Association, Grant/Award Number: 16SDG30450001; National Institute of Allergy and Infectious Diseases, Grant/Award Number: T32 AI095213 ; National Heart, Lung, and Blood Institute, Grant/Award Number: HL153235

Footnotes

CONFLICT OF INTEREST

The authors do not have any conflict of interest.

SUPPORTING INFORMATION

Additional supporting information may be found online in the Supporting Information section.

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