Host-pathogen interactions in cardiovascular infections

UR D-258, Microbes, Evolution, Phylogénie et Infection (MEPHI)
Aix-Marseille Université (AMU)

Team leaders :
DESNUES Benoît (MCF, AMU)
CAMOIN Laurence (PU-PH, AMU/AP-HM)

Members
ABI-RACHED Laurent
ARREGLE Florent
GOURIET Frédérique
HABIB  Gilbert
LEPIDI   Hubert
PONTAROTTI Pierre
STEIN Andreas

Team objectives and projects

The objective of the team “host-pathogen interactions in cardiovascular infections” is to study the host-pathogen crosstalk in patients with cardiovascular infections, notably in infective endocarditis. This will be accomplished using a variety of complementary approaches such as cell and molecular biology, electron microscopy, genetics, and in vitro models of infection. The objective “study of bacteria-host crosstalks” stems from the observation that the initial steps of an infection are critical for the outcome of that infection. Thus, understanding these early events both at the level of the pathogen and of the host is critical for diagnosis, prognosis, and treatment of these infections. In particular, two specific and complementary aims will be developed: 1) a focus on infective endocarditis to improve our understanding of its physiopathology and its diagnosis, and 2) a focus on the role of bacterial determinants in host response using Tropheryma whipplei as a model.

Infective Endocarditis: improving comprehension of physiopathology and diagnosis.

Infective endocarditis (IE) is a bacterial or fungal infection of the endocardium linked to the development of inflammatory and infected fibrino-platelet deposits. This vegetation formation leads to valve destruction. IE occurs both on normal valves and on valves already displaying signs of disease. Despite advances in diagnosis and management, IE is associated with high mortality (Habib et al, 2019).

A breakthrough in IE support is essentially based on three aspects:

  1. Pathophysiology: comprehension of vegetation formation
  2. Diagnosis: identification of pathogens in cases of Blood Culture-Negative Endocarditis (BCNE)
  3. Treatment: Effect of treatment on vegetation structure and on embolic events

Pathophysiology: Comprehension of vegetation formation (Pr L Camoin-Jau, Pr H Lepidi)

The development of IE on a native valve is initiated by a lesion or inflammation of the endothelium of the valve to which the pathogens adhere directly or indirectly. The formation of the vegetation, also called biofilm, is the consequence of permanent interactions between the host and the pathogen. For the host, this occurs through the vascular endothelium, the haemostatic mechanisms, and the immune system. For the pathogen, this involves its adhesion properties, its production of enzymes and toxins, and the formation of the biofilm (Cahill et al, 2016). In vitro studies or animal models do not reproduce these interactions and their consequences in humans. The consequences of comorbidities and long-term treatments, particularly in elderly patients, are not considered in these experimental models (Kouijzer et al, 2022). Yet, a better understanding of IE pathophysiology would help physicians assess the best management and therapeutic strategy for individual patients, leading to improved diagnosis, prognosis, and reduction of the medical costs.

In human, histologic examination of cardiac valves represents the gold standard for the diagnosis of IE at the micrometer scale (Lepidi et al, 2002). Given the complexity and heterogeneity of IE biofilm, the information provided has not led to a better understanding of the pathophysiological mechanisms of IE. Scanning Electron Microscopy (SEM) demonstrated its value to study the adhesion and spatial orientation of bacterial biofilms and the efficacy of antibiotic treatment on cultured biofilms in medical implant trials (Jahanbakhsh S et al, 2020). Focused ion beam scanning electron microscopy was used to study the microenvironment of infected heart valves (Oberbach et al, 2017). However, these methods remain complex to implement.

Zone de Texte: Fig 3We have recently developed an innovative approach for ultrastructural analysis of vegetation based on Scanning Electron Microscopy (SEM) (Hannachi et al, 2020; Baudoin et al 2020). This whole-tissue approach provides nanoscale images of valve tissue in one hour without the need for special stainings, tissue inclusion methods, and ultramicrotomy. It allows the detection and quantification of pathogens and major vegetative components (platelets, fibrin, erythrocytes and leukocytes). The adhesion and spatial localization of the different elements are also analysed. We already demonstrated that the vegetation can be inhomogeneous in its depth (Baudoin et al, 2020) and that the structure of the vegetation varies depending on the bacterial species involved (Hannachi et al, 2020). Because our analysis proved powerful but remains ‘manual’ and based on operator’s analysis, our first objective is to expand further our analysis of IE vegetation ultrastructure by accelerating and standardizing it with the help of atifiical intelligence (AI).  AI approach is rapidly growing both in the fields of microscopy and microbiology (Lopes et al, 2020; Matuszewski et al, 2021). Here, AI will be developed and used to automatically detect and quantify each vegetation component in images from our SEM image database. This will also allow localisation of the bacteria in relation to the blood cells. We hence expect to establish a highly accurate AI pipeline for IE vegetation structure characterization. This innovative approach will make it possible to carry out a structural profile based on the pathogens involved and the state of the patient’s valves (inflamed or damaged valve). These data will improve our understanding of the pathophysiological mechanisms of IE.

This aim is divided in two specific actions:

Standardizing the analysis of the vegetation ultrastructure using SEM and AI

Vegetations will originate from our biobank, which contains material from 300 patients (2012- AOK 1549 34 agreement). Vegetation preparation and microscope settings will follow our published parameters (3). Image acquisition will use a multi-scale (magnification) microscopy strategy with tiled images covering the whole vegetation. Different scales, from x90 to x4000, will be used to image either the vegetation morphology, the cellular or fibrin aggregates, or individual objects of interest which consist in white and red blood cells, fibrin, platelets, and bacteria or fungi. We intend to organize the collected information with a dedicated database. Images will be indexed with metadata regarding patient’s characteristics and acquisition information. The image database will be filled throughout the project duration.

EM being in black and white images, the criteria to discriminate objects of interest will be their shape, dimensions and electron-density. But we aim to automate this tedious task with a deep-learning approach to enable further systematic studies. We intend to define five classes of objects: 1) leukocytes 2) platelets 3) fibrin 4) erythrocytes 5) bacteria or fungi. Images will be annotated by experts (Jean-Pierre Baudoin, Laurence Camoin-Jau). To date, there is little work on EM image analysis with deep learning. Tests are still needed to determine the best architecture and evaluation metrics to be implemented. We intend to choose a suitable network that will be trained to detect and enumerate objects of interest in these large, textured, and low contrast images. To date, Mask-RCNN and Retina-Net are candidates, we plan to assess and adapt to our specific images. As a result, microbe enumeration will be used to build an ultrastructural score for each vegetation, consisting of the abundance of each component and its distribution across the vegetation.

Structural profile of vegetation according to microorganisms and treatment

The vegetation of patients will be systematically analysed by SEM and AI as described. The images will be obtained on distinct areas of the vegetation: area-wise, in the centre and near the valve tissue. For each area examined, we will identify and quantify blood cells (platelets, erythrocytes and leukocytes), amorphous substance and microorganisms in the observed areas. We will compare the ultrastructural features of vegetation’s from IE due to the main pathogens.

We will also compare vegetations from patients treated or not with antithrombotic drugs for the same bacteria.  While platelets and fibrin are major players in the development of IE, anti-thrombotic drugs are not proposed during IE since clinical studies showed conflicting results regarding their use (Anavekar et al 2007; Chan KL et al, 2003). We explain this discrepancy by the lack of discrimination between the different infectious agents involved. As proof, we recently demonstrated that aspirin distinctly modulates in vitro bacteria-platelet aggregation depending on the species, a sine qua non step for the formation of vegetations (Hannachi et al, 2019). Our hypothesis is that structure of vegetation and embolic risk are modulated by aspirin treatment on a bacterial strain-specific way.Our objective will be to collect ultrastructures of IE vegetations differing in i) the microorganism involved; ii) patients with or without anti-platelet drugs for the same microorganism. This will help better understand the pathogen-specific pathophysiology of IE, and in particular the contribution of each cellular component in the development of vegetations. Our study will thus provide precious knowledge for patient’s therapy. This will allow us to evaluate the effect of the nature of the pathogen and of anti-platelet drugs on the structure of vegetations. To the best of our knowledge, there is little data regarding the pathophysiological development and organization of cells in the vegetation, notably the primordial role of platelets and further triggering haemostasis leading to vegetations related to the diversity of microorganisms. In fact, these data mainly originate from animal studies (Garrigos et al, 2019; Liesenborghs et al, 2019), but we will here study patient’s vegetations per se and hence describe bacterial strain-specific and treatment-specific ultrastructural features.

To improve diagnosis (Dr B Desnues)

The diagnosis of IE is often difficult and relies on clinical suspicion, echocardiography, and blood cultures, which have limited sensitivity, especially after medication with antimicrobial agents or when fastidious, slow-growing bacteria, zoonotic bacteria or fungi are involved (Habib et al., 2015). One objective of the team is to define reliable diagnostic and prognostic markers for IE by characterizing, using RNAseq, the metatranscriptomes of heart valves from patients with definite and suspected IE and by validating pathogen and host-derived markers through comprehensive molecular screening along with cellular and animal models.  This aim is divided in three parts:

Link the host response to a documented IE etiologic agent

By analyzing the metatranscriptome of a selected and documented cohort of positive blood culture heart valves using RNA-seq in order to define if the transcriptional profile of the host is pathogen-specific and if the transcriptional signature of the causative pathogen can be detected.

Establish an infectious diagnosis to suspected IE/detect emerging pathogens

By comparing the transcriptional profiles of valves from suspected blood culture-negative IE with the transcriptional profiles defined in action a) and those from non-infective endocarditis.

Validate host and pathogen-derived markers retrospectively

By targeted molecular analysis (qRT-PCR) on a larger cohort. Importantly, RT-PCR systems will also be tested on peripheral blood from the corresponding patients when available to evaluate the systemic character of the putative marker. We will also design cellular assays to validate those markers and eventually to clarify the causation of putative emerging IE.

This specific aim will benefit from the strong and complementary expertise of Pr Gilbert HABIB, cardiologist, and our non-academic collaborator Dr Julien PAGANINI and the XEGEN company. Importantly, this will likely also encourage collaborations with industrials, especially those involved in synthetic valve development in order to prevent risks of IE.

Define the role of bacterial determinants in host response

Another important aim of our project is to understand how the host response towards a given pathogen depends on pathogen determinants.

Host response to intracellular pathogens (Dr B Desnues, Dr L Abi-Rached, Dr P Pontarotti)

We will focus on Tropheryma whipplei, a bacterial pathogen for which the team has a strong and internationally-recognized expertise. T. whipplei is an intracellular bacterium responsible for a wide range of clinical entities encompassing chronic systemic disease (classical Whipple’s disease), chronic localized infections, acute infections and asymptomatic carriage (Boumaza et al, 2022). Accumulating data suggest that T. whipplei evolved strategies to overcome innate immune responses and escape macrophage microbicidal activity (Boumaza et al, 2022). In addition, in patients with chronic T. whipplei infections, histological examination of lesions reveals confluent areas of foamy macrophages strongly colored by periodic acid-Schiff (PAS) staining, containing numerous bacteria and representing the hallmark of the disease.

Whipple’s disease and T. whipplei infections: understanding susceptibility

Until recently, T. whipplei was considered an uncommon microorganism that rarely caused disease. However, evidence now suggests that T. whipplei is ubiquitous and responsible for a large clinical spectrum of infections. Classical Whipple’s disease typically affects middle-aged Caucasian males with a mean age at diagnosis of 55 years (Lagier et al, 2010). Chronic infection of extraintestinal organs can also occur without clinical and histological signs of gastrointestinal involvement (so-called chronic focal/localized infections) (Boumaza et al, 2022) and lead to blood-culture negative endocarditis, encephalitis, or uveitis (Lagier et al, 2010). T. whipplei also causes acute infections with symptoms such as acute gastroenteritis, pneumonia or fever. Finally, asymptomatic carriage, which probably does not lead to higher incidence of Whipple’s disease, was described in the duodenum (0.5% of the general population) and in the stools or the saliva (2-4% in the general population). These epidemiological data raise two main questions: are the different clinical manifestations of T. whipplei dependent on 1) the host (tissular-tropic susceptibility factors) or 2) the bacteria (isolates with different tissular tropisms) and, in particular, is there a genetic susceptibility for T. whipplei infections? In order to answer these two questions, we will address the following actions:

i) Taking advantage of the IHU sample collection to analyze and compare the genomes of various clinical isolates from various specimens (duodenal biopsy, cardiac valve, cerebrospinal fluid, articular fluid, lymph node, muscle, blood, saliva, and cutaneous biopsy) obtained from patients with several conditions due to T. whipplei carriage, acute infections or chronic systemic infections and chronic localized infections. These clinical isolates will be sequenced by NGS and pan-genome analysis of these clinical isolates will provide information regarding minimal core genome (genes present in all the strains), accessory genes (genes present at least in two but not all strains), and unique genes (genes present in only one strain). This presence-absence study will reveal whether specific genes are associated with disease manifestation or tissue tropism but will also provide information regarding T. whipplei lifestyle (sympatry with numerous gene exchange or allopatry with limited gene exchange). Should this analysis fail to reveal clear patterns, a more detailed one will be performed to define the distribution of genetic variants in the core genome and if this variation can explain the phenotypic differences between clinical isolates. This part will benefit from the expertise of Dr Pierre PONTAROTTI in comparative genomics and a non-academic collaboration with Dr Julien PAGANINI and Xegen who are specialized in bioinformatics and NGS/RNA seq analysis.

ii) Several observations support this genetic hypothesis: the strong predominance of cases of Whipple’s disease in Caucasain men despite carriage of the bacterium being much higher in Africa, the existence of familial cases (Gross et al, 1959; Dykman et al, 1999 and Ponz de Leon et al, 2006), and the lifelong susceptibility to T. whipplei infection of Whipple’s disease patients (Lagier et al, 2011). Similarly, interleukin levels of IL-12 produced by macrophages and interferon (IFN-) gamma by T lymphocytes are reduced in patients with Whipple’s disease and this phenotype extends to their family members (Marth et al, 1997). Consistent with a genetic origin, an association between Whipple’s disease and HLA-B27 was reported (Feurle et al, 1979), and HLA-DRB1*13 and DQB1*06 alleles are more frequently found in patients with Whipple’s disease than in asymptomatic carriers (Martinetti et al, 2009). Finally, an autosomal dominant IRF4 deficiency was proposed to underlie Whipple’s disease by haploinsufficiency, with age-dependent incomplete penetrance (Guerin et al, 2018). However, no IRF4 mutations could be found in another cohort, suggesting that other susceptibility loci may be involved. The analysis of patient cohort with T. whipplei infections will be performed by Dr Laurent ABI-RACHED, who has an extensive knowledge in the analysis of immune polymorphisms. RNA-seq analysis on isolated PBMCs/macrophages will be performed on individual immune genes and multigenic families to define coding sequence variation, expression levels or transcript structures that correlate with susceptibility to Whipple’s disease.

Understanding how T. whipplei reaches the mucosa.

First encounter with T. whipplei likely occurs in the infancy and causes mild, self resolutive gastroenteritis. Yet, in some predisposed individuals it causes the classical pathology of Whipple’s disease. In all cases, T. whipplei is thought to enter the body by the small intestine, where it infects mucosal antigen presenting cells (macrophages and dendritic cells) to replicate. However, how the bacteria pass the intestinal epithelial barrier is currently unknown. Similarly, the direct trophic effects of TW on exposed intestinal epithelia are unknown. The objective of this aim is to explore (i) the ability of T. whipplei to pass through the epithelial layer autonomously, (ii) the pathway T. whipplei utilizes to pass the epithelial barrier, and (iii) the capacity of T. whipplei to modulate the integrity of the epithelial barrier. To conduct this analysis, several epithelial cell lines, such as Caco-2, HT29, and T84 will be differentiated to evaluate the effect of T. whipplei on transepithelial resistance and permeability. Apoptosis induction will be also monitored as well as cocultures with macrophages. These data will thus address how T. whipplei translocates into the mucosa.

Understanding the cellular and molecular events of T. whipplei – macrophage interaction.

Although several pieces were recently assembled in the puzzle of T. whipplei infections, the initial steps of the interaction between T. whipplei and macrophages remain largely unknown. We thus propose a dual approach based on cell and molecular biology to address this question.

First, we aim to simultaneously define both host and bacterial gene expression using high-throughput-based transcriptomic technology (dual RNA-seq) at different stages of the infection. This action will benefit from the expertise of Dr Pierre PONTAROTTI in comparative genomics and a non-academic collaboration with Dr Julien PAGANINI and Xegen who are specialized in bioinformatics and NGS/RNA-seq analysis. Differential expression and functional analyses will be performed to identify gene sets modulated during infection. All classes of coding and noncoding transcripts induced or repressed during infection will be targeted, including small noncoding RNA. Finally, network inference approaches will be used to infer links between genes and predict potentially causal interactions. Taken together, these analyses will lead to a better understanding of the physiological changes in the host and bacteria throughout the different stages of the infection.

Second, we aim to characterize the formation of the replicative niche of T. whipplei inside macrophages. Indeed, to counter invasion, xenophagy or LC3-associated phagocytosis is activated at multiple levels to restrict the proliferation of intracellular bacteria and subsequent degradation. However, intracellular bacteria can counter xenophagy by regulating the pattern-recognition receptors, thwarting autophagosome formations, forming bacteria-contained vacuoles to camouflage lysosomes as well as directly altering lysosomal functions. Preliminary experiments suggest that macrophages internalize T. whipplei in a LC-3-dependent pathway and that T. whipplei inhibits later steps of autophagy, thereby favoring its survival by replicating in a vacuole which does not fuse with lyzosomes. Our objective here is to define the chronology of these events and to highlight the potential bacterial effectors involved.

Deciphering the structure and the role of T. whipplei surface glycoproteins

Finally, we aim to decipher the role of T. whipplei surface-associated glycoproteins in shaping macrophage responses and in the pathophysiology of the infection. As T. whipplei induces an alternative macrophage (M2) polarization that favors tolerance, it is important to address to which extent those glycoproteins are involved in the macrophage activation pattern. This action will benefit from strong academic collaborations with world-leading experts in glycobiology, including Dr Bernard HENRISSAT (AFMB, Architecture et Fonction des Macromolécules Biologiques, Marseille), Pr Elisabeth ELASS-ROCHARD and Dr ANNE HARDUIN-LEPERS (UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, Lille). T. whipplei-associated glycoprotein characterization will be addressed by first obtaining a global glycomic profiling using a lectin microarray. Next, T. whipplei glycoproteins will be purified and the protein moieties will be identified by mass spectrometry (MS) while the glycans released from proteins will be analysed by liquid chromatography (LC)-MS. These results will allow the precise characterization of T. whipplei glycoproteins, and by confronting the structure of the glycoconjugates to the genetic glycosylation potential of T. whipplei, we will be able to determine whether those modifications are bacteria-or host-derived.

Platelets and Extracellular pathogens

In addition to their role in haemostasis, platelets play a major role in the anti-infective response and in the regulation of the inflammatory response (Koupenova et al, 2018). This anti-infective defence role of platelets was demonstrated by their ability to interact and activate in the presence of many classes of pathogens. Many cell-surface receptors of platelets are involved in the interaction with bacteria, such as TLRs, the PAF receptor, FcγRIIA or GPIbα (Yeaman et al, 2014). Bacteria can interact with one or more of these receptors and induce platelet activation that can lead to both deleterious phenomena for the host, such as thrombosis or deregulated inflammation, or beneficial ones, with a demonstrated bactericidal effect of platelets on certain bacterial strains (Hannachi et al, 2020).

Bidirectional interactions between platelets and pathogens

Microbiologically, staphylococci, streptococci and enterococci are the top three etiologic agents of IE (Liesman et al, 2017); E. faecalis is responsible for ∼10% of all infective endocarditis cases (Fernández-Hidalgo et al, 2020). Notably, the aetiology of infective endocarditis was reported to be shifting towards an increased prevalence of enterococci as the causative agent in several recent regional reports. We are interested in the bidirectional interaction of platelets with these three species, namely the ability of these bacterial species to induce platelet activation and consequently their sensitivity to the bactericidal activity of platelets.

Platelet activation induced by bacteria species

We previously demonstrated that platelets activate differently depending on the bacterial species involved (Hannachi et al, 2020, Hannachi et al, 2020). This preliminary work demonstrated that enterococcus strains induce platelet activation and aggregation in a variety of ways depending on the strain and patient. Our objective is to establish the phenotypes of the strains responsible for platelet activation and to study the intra-platelet signalling pathways mobilized.

While platelets are fragments of megakaryocytes cytoplasm, they contain Tissue Factor RNA. Expression of Tissue Factor induces coagulation and also fibrin formation. Our objective is to characterize this phenomenon according to the strains and to study the signalling pathways involved in order to propose antithrombotic drugs.

In order to highlight the variability of patient response, the analysis of patient cohorts with enterococcus IE will be performed by Dr Laurent ABI-RACHED, who has an extensive knowledge in the analysis of immune polymorphisms. The RNA-seq analysis will be performed as described in B.1.a on individual genes and multigenic families to define coding sequence variation, expression levels or transcript structures that correlate with the different phenotypes of platelet activation and aggregation.

Platelet bactericidal activity

Platelet activation induces granule secretion and secretion of Platelet Microbicidal Peptides (PMP). We showed that S. aureus are sensitive to the bactericidal activity of platelets (Hannachi et al, 2020). Surprisingly, limited data are available regarding the sensitivity of streptococcus and enterococcus to this phenomenon. We will evaluate the capacity of platelets to inhibit platelet growth and/or to kill streptococcus and enterococcus. The evasion of platelet-mediated innate immunity may explain bacteria virulence and needs to be analyzed.

 Analysis of PMP by proteomic will allow to determine the PMP implied according to species.

Effect of antithrombotic drugs on platelets-bacteria interactions

Anti-platelet therapy is a promising potential adjuvant therapy in IE prevention. Given the availability of anti-platelet drugs, pharmacologic targeting of platelet function represents an attractive approach to mitigate platelet-assisted excessive inflammation that contributes to IE progression. We previously showed that platelet activation induced by S. aureus is partially inhibited by antiplatelet drugs with different responses depending on the drug tested (Hannachi et al, 2020).

To extend our findings, we propose to study the effect of antiplatelet treatments on the interactions between streptococcus and enterococcus species. This work will be compared with the study carried out by electron microscopy on the vegetations of patients with IE treated long-term with antiplatelet drugs. In addition to our work on antiplatelets, we will look at the effect of direct oral anticoagulants. Indeed,dabigatran, a thrombin inhibitor, was extensively studied in preclinical IE in vivo studies with promising results (Lerche et al., 2021). Direct thrombin inhibitors are safe in patients with S. aureus bacteremia (Peetermans et al., 2018) and lower the incidence of developing S. aureus bacteremia in patients with atrial fibrillation on anticoagulants (Butt et al., 2021).

Conclusions

This project integrates strong and complementary expertise in the field of pathophysiology, cell biology, microbiology, haemostasis, electron microscopy, OMICS data analysis, and bioinformatics. All infrastructures, platforms, servers and facilities available at IHU Méditerranée Infection will also facilitate the success of this ambitious project.

Publications

Last 5 years on Pubmed

References

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