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The invasive pathogen Yersinia pestis disrupts host blood vasculature to spread and provoke hemorrhages

['Guillain Mikaty', 'Institut Pasteur', 'Yersinia Research Unit', 'Paris', 'Héloïse Coullon', 'Laurence Fiette', 'Unité D Histopathologie Humaine Et Modèles Animaux', 'Javier Pizarro-Cerdá', 'Elisabeth Carniel']

Date: 2021-10

Abstract Yersinia pestis is a powerful pathogen with a rare invasive capacity. After a flea bite, the plague bacillus can reach the bloodstream in a matter of days giving way to invade the whole organism reaching all organs and provoking disseminated hemorrhages. However, the mechanisms used by this bacterium to cross and disrupt the endothelial vascular barrier remain poorly understood. In this study, an innovative model of in vivo infection was used to focus on the interaction between Y. pestis and its host vascular system. In the draining lymph nodes and in secondary organs, bacteria provoked the porosity and disruption of blood vessels. An in vitro model of endothelial barrier showed a role in this phenotype for the pYV/pCD1 plasmid that carries a Type Three Secretion System. This work supports that the pYV/pCD1 plasmid is responsible for the powerful tissue invasiveness capacity of the plague bacillus and the hemorrhagic features of plague.

Author summary The plague bacillus, Yersinia pestis, is a powerful pathogen with a rare invasive capacity and is among the few bacteria capable to provoke disseminated hemorrhages. However, the mechanisms used by this bacterium to cross and disrupt the endothelial vascular barrier remain poorly understood. Recent technical progress in microscopy, associated with the use of original fluorescent mutant in mice, allowed us to develop an innovative model of infection in vivo. This model permitted to look directly into the interaction between Y. pestis and its host vascular system, in 3D reconstructed tissues without physical alteration. We were able to observe the degradation of blood vessels in the draining lymph nodes and to visualize the spreading of the bacteria into secondary organs directly through the vascular barrier. Classical in vitro experiments validated the in vivo observation and demonstrated the role of some of the bacterial components in this phenotype. This work shows an unprecedented visualization of the pathogenesis of Y. pestis and decipher part of the powerful invasiveness capacity of the plague bacillus and the hemorrhagic features of plague.

Citation: Mikaty G, Coullon H, Fiette L, Pizarro-Cerdá J, Carniel E (2021) The invasive pathogen Yersinia pestis disrupts host blood vasculature to spread and provoke hemorrhages. PLoS Negl Trop Dis 15(10): e0009832. https://doi.org/10.1371/journal.pntd.0009832 Editor: R. Manjunatha Kini, National University of Singapore, SINGAPORE Received: May 17, 2021; Accepted: September 22, 2021; Published: October 5, 2021 Copyright: © 2021 Mikaty et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the manuscript and its Supporting Information files. Funding: GM received grants from the CEA (NRBC project #17.1). https://www.cea.fr/Pages/domaines-recherche/defense-securite/recherches-CEA-programme-NRBC-E.aspx The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Plague is an infection caused by the invasive Gram-negative bacillus Yersinia pestis. It is among the most dramatic bacterial diseases in human history [1]. In spite of its disappearance in most developed countries, plague still represents a significant public health problem in many regions of the world [2]. Madagascar is currently the most active plague focus worldwide, with hundreds of human cases every year and regular epidemics [3]. Since the 1990’s, the disease has also re-emerged in countries where it was thought to be extinct [4–6]. Furthermore, the discovery of natural isolates of Y. pestis carrying antibiotic-resistance plasmids [7,8] associated with the possibility of using Y. pestis as a bioweapon in the international context of terrorism is also of global concern. Bubonic plague, the most common clinical presentation in humans and the usual form of plague in the rodent reservoir, occurs after an infectious fleabite. Studies in animal models have shown that Y. pestis may remain for various periods of time at the site of inoculation in the dermis and multiply locally. Afterwards the bacteria are drained through the lymphatic flux to the proximal lymph node where they form the pathognomonic bubo, and then to the secondary ipsilateral lymph node [9–11]. Inside the lymph nodes, Y. pestis enters through the subcapsular sinus, where the bacteria replicate and spread within the sinus. Bacteria spread into the cortex where they multiply, provoking the recruitment of numerous polymorphonuclear leukocytes. If the innate immune system of the lymph node is strong enough to contain the bacteria, the bubo suppurates and the patient recovers. However, in most instances the bacteria overpower the innate immune system of the lymph node and spread systemically [12,13]. The time elapsed between the colonization of the lymph node and the fatal outcome is very short (2.2 days on average) in the mouse experimental model [10]. Several hypotheses have been proposed for the mechanism used by Y. pestis to enter the bloodstream: (i) an invasive process by active degradation of the blood vessels of the node [11], (ii) a release of the bacteria carried by the lymphatic vessels into the blood through the subclavian vein, or (iii) a carriage of Y. pestis inside leukocytes from the lymph to the blood [14]. Once in the blood, bacteria are filtered by secondary lymphoid organs (spleen and liver), until the filtering capacity of these organs is overwhelmed, allowing the bacteria to spread and cause a severe and terminal septicemia, sometimes associated with internal and external bleedings. Massive and diffuse hemorrhages in all tissues including the lymph nodes, have been a striking feature of post-mortem pathological examination of human plague victims [13,15]. Comparable hemorrhages were also reported in wild animals that succumbed to plague [16,17]. This feature of plague pathogenesis is experimentally reproducible in various animal models [11,12,18,19]. A classically accepted view is that a Disseminated Intravascular Coagulopathy (DIC) occurs during septicemic stages and provokes these hemorrhages [18,20,21]. However, DIC usually occurs late in the pathogenesis of Gram-negative bacteria whereas hemorrhages are observable as early as day 3 post-infection with Y. pestis. Thus suggesting that Y. pestis has the ability to alter blood vessel integrity independently of coagulation defect. Y. pestis express various virulence factors that play important roles during the invasion and colonization of its hosts. The high-pathogenicity island, or HPI, carries essential virulence genes involved in iron acquisition and is encoded on the chromosome [22]. Three plasmids carry specific virulence factors [23–24]: i) The pPla plasmid (also known as pPCP1) carries notably the plasminogen activator Pla, an adhesin with enzymatic activities capable of converting plasminogen to plasmin, thus degrading extracellular matrix and fibrin clots in vivo; ii) the pMT plasmid (also known as pFra) of Y. pestis carries the caf 1 gene that encodes for the F1 pseudocapsule, involved in the resistance to phagocytosis [25]; iii) the pYV/pCD1 plasmid carries genes necessary to synthetize a Type Three Secretion System (TTSS) that allows the injection of effectors (Yops) inside the host cell cytosol [26]. These three plasmids are associated with Y. pestis virulence. The pYV/pCD1 plasmid is shared by and essential for the virulence of the three pathogenic Yersinia species, Y. pestis, Y. pseudotuberculosis and Y. enterocolitica [27]. They might be involved in the powerful invasiveness of Y. pestis. Although the capacity of Y. pestis to alter the vascular barriers may be important during the pathological process, the interactions of the pathogen with endothelial cells remain mainly unexplored. The aim of this study was, using in vitro and in vivo models of infection, to get insight into the processes that allow Y. pestis to cross the blood vessel barrier and ultimately cause hemorrhages. The results show that Y. pestis can degrade blood vasculature within the draining lymph node in vivo, certainly causing the internal hemorrhages frequently observable. Once in the bloodstream, bacteria can also spread systematically and cross the vascular barrier from the lumen to the organs, eventually degrading the tissues in the secondary organs. The in vitro model of infection of cellular vascular barrier highlights the central role of the pYV/pCD1 plasmid in this phenomenon.

Materials and methods Ethics statement All animals were housed in a level 3 animal facility accredited by the French Ministry of Agriculture (accreditation B 75 15–01), and were infected in compliance with French and European regulations (EC Directive 86/609, French Law 2001–486), following the approved protocol CETEA 2014-0025/MESR 008223 by the internal Institut Pasteur ethic board. For infection of Flk-1GFP/+ mice, death of animals was never intended as outcome of the experiments and no mice died before planned sacrifice. For the LD50 measurement of the Y. pestis CO92 pFU96+ strain, death of animal was the planned outcome. Humane endpoint were defined and animals were monitored twice a day; animals appearing evidently moribund (apathetic, shivering, cold, etc.) were immediately sacrificed. EC and GM were trained by the Institut Pasteur internal training for handling and care of animals and for animal experimentation. Bacteria and culture The Y. pestis strains used were CO92 wild type [28] and its pYV/pCD1-cured and Δcaf derivatives [29]; 6/69 wild type and its pPla-cured derivatives [30,31]. Plasmid pFU96 [32] that confers red-fluorescence was kindly provided by P. Dersch (department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany) and was introduced into Y. pestis CO92 by electroporation. For animal experiments, bacteria were cultured at 28°C for 36h on Luria Bertani agar plates supplemented with 0.002% (w/v) hemin (LBH). For cell infection, bacteria were grown on LBH plates at 28°C for 24h, and at 37°C for another 12h. All experiments with live Y. pestis were performed in a biosafety level 3 laboratory. Animal experiments Flk-1GFP/+ mice [33] exhibiting a bright GFP signal in all endothelial cells due to the insertion of the gene encoding green fluorescent protein (GFP) into the VEGF receptor-2 gene locus, were kindly provided by Alexander Medvinsky (Institute for Stem Cell Research, University of Edinburgh, UK). For infection, 100 μl of 5x103 cfu/ml bacterial suspensions were injected subcutaneously into the right lateral ventral region. The LD50 of the Y. pestis CO92 pFU96+ strain was measured using six-week-old OF1 female mice (Charles River Laboratory) according to the method of Reed and Muench [34]. Cell culture & infection Human Dermal Microvascular Endothelial Cells (Promocell) were grown in Endothelial Cell Medium MV2 with supplements (Promocell) at 37°C in 5% CO2. Five days prior to infection, cells were plated on glass coverslips in 24-well plates (for immunofluorescence experiments) or on Transwells (0.4 or 3 μm filters) coated with Type-I Collagen (R&D System) at a density of 0.25x105 cells/well. Continuous cell monolayers (≈5x105 cells/well) were infected at a Multiplicity of Infection (MOI) of 100 for 30 min to allow bacterial adhesion. After washing with PBS and addition of fresh medium to the monolayer, the incubation was continued for at least 2h. The MOI and the timing were chosen following preliminary experiments in which a serial increasing MOI was used to infect HDMEC cells (1; 10; 50; 100; 1000) for various times (30’, 60’, 90, 120’, 240’). In all conditions, the outcome was the same, only the percentage of rounding was varying depending on the MOI and time. Infection too long or with a MOI too strong resulted in detachment and/or death of the cells. MOI 100 for 150 minutes was found to be optimal for the observation of cell rounding with limited cell death/detachment, and was subsequently selected for further experiments. In a first set of permeability assays, 1 mg/ml of 4 kDa FITC-Dextran (Sigma aldrich) was added to the upper chamber of 0.4 μm Transwells covered with HDMEC that were either untreated (control) or infected for 2.5h. Every 20 min, 100 μl samples from the lower chamber were taken to measure optical density at 485 nm and 530 nm for FITC excitation and emission respectively. Fluorescence value was converted into protein mass using the fluorescence measured for a standard curve of 4 kDa FITC-Dextran. In another set of permeability assays to measure the capacity of Y. pestis to translocate through the cell monolayer, 3 μm Transwells were used. The bacteria in the upper chamber were not removed after 30 min, and 100 μl samples were taken from the lower chamber after 3.5h and 4.5h to enumerate cfu. Bacterial filtrates were obtained from filtration (through at 0.22 μm filter) of suspensions of 5x107 bacteria cultured in cell culture medium at 28°C or 37°C for 3 hours. Bacterial sonicates were obtained from sonication of 109 bacteria cultured on Agar plate at 28°C or 37°C and resuspended in cold saline added with protease inhibitors (Roche, Complete) and PMSF (Sigma). Sonicates were filtrated through 0.22 μm filters. Cells (2x105 HDMEC) were incubated for 5 hours at 37°C with the equivalent of 5x107 bacterial filtrate or sonicate. Confocal microscopy, histology and immunofluorescence microscopy Right inguinal lymph nodes were recovered as draining lymph node and left inguinal lymph nodes as secondary infected organs. Lymph nodes and spleen were fixed in 4% neutral buffered paraformaldehyde (PFA) for 48h, sliced with a 200 μm-thick setting using a vibratome (Leica VT 1200S), and mounted with Vectashield mounting medium (Vector Laboratories). Slides were observed and imaged with a spinning-disc Cellvoyager CV1000 confocal system (Yokagawa) at the Imagopole of the Center for Innovation & Technological Research from the Institut Pasteur. For histopathological examination, non-infected inguinal lymph nodes were taken and fixed in 4% neutral buffered formalin for 48h, and embedded in paraffin. Four μm tissue sections were stained with hematoxylin-eosin. Histology slides were examined through an Eclipse 5Oi Nikon microscope equipped with a DSRi1 camera (Nikon). In vitro-infected cells were fixed in 4% PFA at specified times and exposed to the following primary antibodies for 1h: mouse anti-F1 monoclonal antibody (1:500; B18-1, [35]); rabbit anti-Yersinia polyclonal antiserum (1:1000; [12]), mouse anti-human VE-Cadherin monoclonal antibody (1:100; BV9, Abcam). Afterward, they were washed 3 times with PBS and incubated with the following secondary antibodies for 1h: Alexa Fluor 488 goat anti-mouse (1:500; Invitrogen) or anti-rabbit (1:1000; Invitrogen) IgG; rhodamin-phalloidin (1:200; Invitrogen), and DAPI (1:10.000, Interchim). After three washings with PBS, stained cells were mounted in moviol and observed under a confocal microscope LSM700 upright (Zeiss) coupled to a color camera. The average number of round cells per field was determined by counting the number of cells in at least three different fields per condition for each experiment. Cell fields were chosen randomly using the DAPI channel, then the field was switched to green and the presence of holes around the cell was enumerated. All the in vitro experiments were repeated independently at least three times. Images were processed and analyzed using Photoshop (Adobe) for immunofluorescence experiments and FIJI (ImageJ) for confocal experiments. No non-linear alterations were brought to the images.

Discussion In order to study the interaction between Y. pestis and its host vasculature directly in vivo, a new approach was developed using a mouse strain expressing GFP-tagged blood vessels infected with a RFP-tagged Y. pestis. This technology permitted an innovative, qualitative observation of infection and of the interaction between Y. pestis and its host at the organ level (using spinning-disc confocal microscopy) and at the cellular level (using confocal microscopy). The use of a spinning-disc confocal microscopy allowed analyzing the entire lymphoid organ (3x1.5 millimeters long and up to a millimeter of thickness) with a minimal transformation and alteration of the sample due to staining or treatment. In this study, this approach showed the previously undocumented degradation of the blood vessels by Y. pestis during infection. We propose that the degradation of blood vessels is an active mechanism caused directly by Y. pestis as observed in vitro. An alternative explanation would be an indirect effect of Y. pestis on blood vasculature through the inflammatory response to the infection. Massive recruitment of Polynuclear Neutrophils is known to provoke an increase in vascular permeability that could lead to bleeding in some cases [36]. However, neutrophils recruitment does not induce a complete physical degradation of the vessels themselves, such as the one observed in the infected lymph nodes. Additionally, an inflammatory process would have a distant effect, nevertheless, Fig 1 shows that only blood vessels in close proximity with bacteria seem to disappear, suggesting that it is the direct contact of Y. pestis with endothelial cells that provokes the degradation of the vessels, as seen in vitro, and thus excluding a major role of the inflammatory process in this phenomenon. This point is of importance since the mechanism used to enter the bloodstream remains controversial. Previous work by Nahm and colleagues showed that the variable timing between the subcutaneous infection of Y. pestis and the outcome is due to the time to reach the lymph node [10]. Once in the draining lymph node, Y. pestis quickly spread to other internal organs, the infection is rapid and often fatal. Thus suggesting that lymph nodes are the entry door to blood circulation. The main argument against the hypothesis for a direct entry of the bacillus into the bloodstream was the absence of proof that Y. pestis enters in contact with vessels and could cross the vascular barrier. This work demonstrates that both events take place in vivo in the bubo. Though it does not invalidate other hypotheses, the data presented strongly support for an active and direct entry of Y. pestis, in a pYV/pCD1 dependent manner, into the blood circulation and explains the invasiveness of this pathogen. The increased blood vessel permeability allows the crossing of bacteria, which could also explain systemic hemorrhages associated with plague. Past studies suggested that hemorrhages due to Y. pestis were the consequences of a DIC provoked by the toxicity of the bacterial LPS [18,20]. However, this work suggests a more direct and faster effect of the bacteria on blood vasculature. Y. pestis can breach the blood barrier in vitro and in vivo inducing an increased permeability that could provoke internal bleedings. Among the virulence factors carried by Y. pestis, the pYV/pCD1 plasmid is one genetic element crucial to the pathogenicity [25]. This ≈70 kb plasmid encodes a TTSS that mediates the injection of molecular effectors, the Yop proteins, directly into the cell cytoplasm. Inside the cells, these Yops disorganize the actin cytoskeleton (thus preventing phagocytosis), inhibit signaling cascades involved in the early innate response, and induce apoptosis of the target cell [26,37,38]. We propose that the pYV/pCD1 plasmid is responsible for the disruption of blood vessels, probably through a direct action of the Yop effectors injected into the cytoplasm of endothelial cells. The fact that neither the bacterial sonicate nor the bacterial supernatant can produce the rounding phenotype observed during infection suggests that the Yop effectors themselves cannot act from outside the eukaryotic cells. We assume that the injection into the cytoplasm is necessary for inducing the rounding. The YopT and YopE effectors are good candidates as they have been demonstrated to have a rounding capacity on Hela cells [39,40,41]. The TTSS has been showed to be crucial in Y. pestis pathogenicity and a deletion mutant of pYV/pCD1 is virtually non-pathogenic. In particular, a pYV/pCD1- strain injected by IV route is almost non-pathogenic. We propose that the pYV/pCD1 plasmid encoding the TTSS is responsible for the impressive invasiveness of Y. pestis and for its hemorrhagic features.

Acknowledgments The authors would like to thank Petra Dersch from the department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, in Germany, for providing the plasmid pFU96 and Anne Derbise, in the Yersinia research unit for her benevolent assistance in constructing the RFP-CO92 strain. We are very grateful to Fabrice Chrétien and Claire Latroche from the Histopathology and Animal Models Unit at the Institut Pasteur, Paris, France, for providing the Flk1-GFP mice. We would like to thank Christian Demeure for his critical reading of the manuscript and with Sofia Filali from in the Yersinia research Unit for their helpful support with animal experiments over the years.

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