Yersinia pestis and Plague: some knowns and unknowns.

Since its first identification in 1894 during the third pandemic in Hong Kong, there has been significant progress of understanding the lifestyle of Yersinia pestis, the pathogen that is responsible for plague. Although we now have some understanding of the pathogen's physiology, genetics, genomics, evolution, gene regulation, pathogenesis and immunity, there are many unknown aspects of the pathogen and its disease development. Here, we focus on some of the knowns and unknowns relating to Y. pestis and plague. We notably focus on some key Y. pestis physiological and virulence traits that are important for its mammal-flea-mammal life cycle but also its emergence from the enteropathogen Yersinia pseudotuberculosis. Some aspects of the genetic diversity of Y. pestis, the distribution and ecology of plague as well as the medical countermeasures to protect our population are also provided. Lastly, we present some biosafety and biosecurity information related to Y. pestis and plague.

Preclinical safety and efficacy characterization of an LpxC inhibitor against Gram-negative pathogens.

The UDP-3-O-(R-3-hydroxyacyl)-N-acetylglucosamine deacetylase LpxC is an essential enzyme in the biosynthesis of lipid A, the outer membrane anchor of lipopolysaccharide and lipooligosaccharide in Gram-negative bacteria. The development of LpxC-targeting antibiotics toward clinical therapeutics has been hindered by the limited antibiotic profile of reported non-hydroxamate inhibitors and unexpected cardiovascular toxicity observed in certain hydroxamate and non-hydroxamate-based inhibitors. Here, we report the preclinical characterization of a slow, tight-binding LpxC inhibitor, LPC-233, with low picomolar affinity. The compound is a rapid bactericidal antibiotic, unaffected by established resistance mechanisms to commercial antibiotics, and displays outstanding activity against a wide range of Gram-negative clinical isolates in vitro. It is orally bioavailable and efficiently eliminates infections caused by susceptible and multidrug-resistant Gram-negative bacterial pathogens in murine soft tissue, sepsis, and urinary tract infection models. It displays exceptional in vitro and in vivo safety profiles, with no detectable adverse cardiovascular toxicity in dogs at 100 milligrams per kilogram. These results establish the feasibility of developing oral LpxC-targeting antibiotics for clinical applications.

A Widefield Light Microscopy-Based Approach Provides Further Insights into the Colonization of the Flea Proventriculus by Yersinia pestis.

Yersinia pestis (the agent of flea-borne plague) must obstruct the flea's proventriculus to maintain transmission to a mammalian host. To this end, Y. pestis must consolidate a mass that entrapped Y. pestis within the proventriculus very early after its ingestion. We developed a semiautomated fluorescent image analysis method and used it to monitor and compare colonization of the flea proventriculus by a fully competent flea-blocking Y. pestis strain, a partially competent strain, and a noncompetent strain. Our data suggested that flea blockage results primarily from the replication of Y. pestis trapped in the anterior half of the proventriculus. However, consolidation of the bacteria-entrapping mass and colonization of the entire proventricular lumen increased the likelihood of flea blockage. The data also showed that consolidation of the bacterial mass is not a prerequisite for colonization of the proventriculus but allowed Y. pestis to maintain itself in a large flea population for an extended period of time. Taken as the whole, the data suggest that a strategy targeting bacterial mass consolidation could significantly reduce the likelihood of Y. pestis being transmitted by fleas (due to gut blockage), but also the possibility of using fleas as a long-term reservoir. IMPORTANCE Yersinia pestis (the causative agent of plague) is one of the deadliest bacterial pathogens. It circulates primarily among rodent populations and their fleas. Better knowledge of the mechanisms leading to the flea-borne transmission of Y. pestis is likely to generate strategies for controlling or even eradicating this bacillus. It is known that Y. pestis obstructs the flea's foregut so that the insect starves, frantically bites its mammalian host, and regurgitates Y. pestis at the bite site. Here, we developed a semiautomated fluorescent image analysis method and used it to document and compare foregut colonization and disease progression in fleas infected with a fully competent flea-blocking Y. pestis strain, a partially competent strain, and a noncompetent strain. Overall, our data provided new insights into Y. pestis' obstruction of the proventriculus for transmission but also the ecology of plague.

No evidence for persistent natural plague reservoirs in historical and modern Europe.

Caused by Yersinia pestis, plague ravaged the world through three known pandemics: the First or the Justinianic (6th-8th century); the Second (beginning with the Black Death during c.1338-1353 and lasting until the 19th century); and the Third (which became global in 1894). It is debatable whether Y. pestis persisted in European wildlife reservoirs or was repeatedly introduced from outside Europe (as covered by European Union and the British Isles). Here, we analyze environmental data (soil characteristics and climate) from active Chinese plague reservoirs to assess whether such environmental conditions in Europe had ever supported "natural plague reservoirs". We have used new statistical methods which are validated through predicting the presence of modern plague reservoirs in the western United States. We find no support for persistent natural plague reservoirs in either historical or modern Europe. Two factors make Europe unfavorable for long-term plague reservoirs: 1) Soil texture and biochemistry and 2) low rodent diversity. By comparing rodent communities in Europe with those in China and the United States, we conclude that a lack of suitable host species might be the main reason for the absence of plague reservoirs in Europe today. These findings support the hypothesis that long-term plague reservoirs did not exist in Europe and therefore question the importance of wildlife rodent species as the primary plague hosts in Europe.

What do we know about osmoadaptation of Yersinia pestis?

The plague agent Yersinia pestis mainly spreads among mammalian hosts and their associated fleas. Production of a successful mammal-flea-mammal life cycle implies that Y. pestis senses and responds to distinct cues in both host and vector. Among these cues, osmolarity is a fundamental parameter. The plague bacillus lives in a tightly regulated environment in the mammalian host, while osmolarity fluctuates in the flea gut (300-550 mOsM). Here, we review the mechanisms that enable Y. pestis to perceive fluctuations in osmolarity, as well as genomic plasticity and physiological adaptation of the bacterium to this stress.

Antibiotic Therapy of Plague: A Review.

Plague-a deadly disease caused by the bacterium Yersinia pestis-is still an international public health concern. There are three main clinical forms: bubonic plague, septicemic plague, and pulmonary plague. In all three forms, the symptoms appear suddenly and progress very rapidly. Early antibiotic therapy is essential for countering the disease. Several classes of antibiotics (e.g., tetracyclines, fluoroquinolones, aminoglycosides, sulfonamides, chloramphenicol, rifamycin, and ß-lactams) are active in vitro against the majority of Y. pestis strains and have demonstrated efficacy in various animal models. However, some discrepancies have been reported. Hence, health authorities have approved and recommended several drugs for prophylactic or curative use. Only monotherapy is currently recommended; combination therapy has not shown any benefits in preclinical studies or case reports. Concerns about the emergence of multidrug-resistant strains of Y. pestis have led to the development of new classes of antibiotics and other therapeutics (e.g., LpxC inhibitors, cationic peptides, antivirulence drugs, predatory bacteria, phages, immunotherapy, host-directed therapy, and nutritional immunity). It is difficult to know which of the currently available treatments or therapeutics in development will be most effective for a given form of plague. This is due to the lack of standardization in preclinical studies, conflicting data from case reports, and the small number of clinical trials performed to date.

Interplay between Yersinia pestis and its flea vector in lipoate metabolism.

To thrive, vector-borne pathogens must survive in the vector's gut. How these pathogens successfully exploit this environment in time and space has not been extensively characterized. Using Yersinia pestis (the plague bacillus) and its flea vector, we developed a bioluminescence-based approach and employed it to investigate the mechanisms of pathogenesis at an unprecedented level of detail. Remarkably, lipoylation of metabolic enzymes, via the biosynthesis and salvage of lipoate, increases the Y. pestis transmission rate by fleas. Interestingly, the salvage pathway's lipoate/octanoate ligase LplA enhances the first step in lipoate biosynthesis during foregut colonization but not during midgut colonization. Lastly, Y. pestis primarily uses lipoate provided by digestive proteolysis (presumably as lipoyl peptides) rather than free lipoate in blood, which is quickly depleted by the vector. Thus, spatial and temporal factors dictate the bacterium's lipoylation strategies during an infection, and replenishment of lipoate by digestive proteolysis in the vector might constitute an Achilles' heel that is exploited by pathogens.

Yersinia pestis Plasminogen Activator.

The Gram-negative bacterium Yersinia pestis causes plague, a fatal flea-borne anthropozoonosis, which can progress to aerosol-transmitted pneumonia. Y. pestis overcomes the innate immunity of its host thanks to many pathogenicity factors, including plasminogen activator, Pla. This factor is a broad-spectrum outer membrane protease also acting as adhesin and invasin. Y. pestis uses Pla adhesion and proteolytic capacity to manipulate the fibrinolytic cascade and immune system to produce bacteremia necessary for pathogen transmission via fleabite or aerosols. Because of microevolution, Y. pestis invasiveness has increased significantly after a single amino-acid substitution (I259T) in Pla of one of the oldest Y. pestis phylogenetic groups. This mutation caused a better ability to activate plasminogen. In paradox with its fibrinolytic activity, Pla cleaves and inactivates the tissue factor pathway inhibitor (TFPI), a key inhibitor of the coagulation cascade. This function in the plague remains enigmatic. Pla (or pla) had been used as a specific marker of Y. pestis, but its solitary detection is no longer valid as this gene is present in other species of Enterobacteriaceae. Though recovering hosts generate anti-Pla antibodies, Pla is not a good subunit vaccine. However, its deletion increases the safety of attenuated Y. pestis strains, providing a means to generate a safe live plague vaccine.

A refined model of how Yersinia pestis produces a transmissible infection in its flea vector.

In flea-borne plague, blockage of the flea's foregut by Yersinia pestis hastens transmission to the mammalian host. Based on microscopy observations, we first suggest that flea blockage results from primary infection of the foregut and not from midgut colonization. In this model, flea infection is characterized by the recurrent production of a mass that fills the lumen of the proventriculus and encompasses a large number of Y. pestis. This recurrence phase ends when the proventricular cast is hard enough to block blood ingestion. We further showed that ymt (known to be essential for flea infection) is crucial for cast production, whereas the hmsHFRS operon (known to be essential for the formation of the biofilm that blocks the gut) is needed for cast consolidation. By screening a library of mutants (each lacking a locus previously known to be upregulated in the flea gut) for biofilm formation, we found that rpiA is important for flea blockage but not for colonization of the midgut. This locus may initially be required to resist toxic compounds within the proventricular cast. However, once the bacterium has adapted to the flea, rpiA helps to form the biofilm that consolidates the proventricular cast. Lastly, we used genetic techniques to demonstrate that ribose-5-phosphate isomerase activity (due to the recent gain of a second copy of rpiA (y2892)) accentuated blockage but not midgut colonization. It is noteworthy that rpiA is an ancestral gene, hmsHFRS and rpiA2 were acquired by the recent ancestor of Y. pestis, and ymt was acquired by Y. pestis itself. Our present results (i) highlight the physiopathological and molecular mechanisms leading to flea blockage, (ii) show that the role of a gene like rpiA changes in space and in time during an infection, and (iii) emphasize that evolution is a gradual process punctuated by sudden jumps.

Polymorphism in the Yersinia LcrV Antigen Enables Immune Escape From the Protection Conferred by an LcrV-Secreting Lactococcus Lactis in a Pseudotuberculosis Mouse Model.

Yersinioses caused by Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica are significant concerns in human and veterinary health. The link between virulence and the potent LcrV antigen has prompted the latter's selection as a major component of anti-Yersinia vaccines. Here, we report that (i) the group of Yersinia species encompassing Y. pestis and Y. pseudotuberculosis produces at least five different clades of LcrV and (ii) vaccination of mice with an LcrV-secreting Lactococcus lactis only protected against Yersinia strains producing the same LcrV clade as that of used for vaccination. By vaccinating with engineered LcrVs and challenging mice with strains producing either type of LcrV or a LcrV mutated for regions of interest, we highlight key polymorphic residues responsible for the absence of cross-protection. Our results show that an anti-LcrV-based vaccine should contain multiple LcrV clades if protection against the widest possible array of Yersinia strains is sought.

Nutrient depletion may trigger the Yersinia pestis OmpR-EnvZ regulatory system to promote flea-borne plague transmission.

The flea's lumen gut is a poorly documented environment where the agent of flea-borne plague, Yersinia pestis, must replicate to produce a transmissible infection. Here, we report that both the acidic pH and osmolarity of the lumen's contents display simple harmonic oscillations with different periods. Since an acidic pH and osmolarity are two of three known stimuli of the OmpR-EnvZ two-component system in bacteria, we investigated the role and function of this Y. pestis system in fleas. By monitoring the in vivo expression pattern of three OmpR-EnvZ-regulated genes, we concluded that the flea gut environment triggers OmpR-EnvZ. This activation was not, however, correlated with changes in pH and osmolarity but matched the pattern of nutrient depletion (the third known stimulus for OmpR-EnvZ). Lastly, we found that the OmpR-EnvZ and the OmpF porin are needed to produce the biofilm that ultimately obstructs the flea's gut and thus hastens the flea-borne transmission of plague. Taken as a whole, our data suggest that the flea gut is a complex, fluctuating environment in which Y. pestis senses nutrient depletion via OmpR-EnvZ. Once activated, the latter triggers a molecular program (including at least OmpF) that produces the biofilm required for efficient plague transmission.

To block or not to block: The adaptive manipulation of plague transmission.

The ability of the agent of plague, Yersinia pestis, to form a biofilm blocking the gut of the flea has been considered to be a key evolutionary step in maintaining flea-borne transmission. However, blockage decreases dramatically the life expectancy of fleas, challenging the adaptive nature of blockage. Here, we develop an epidemiological model of plague that accounts for its different transmission routes, as well as the within-host competition taking place between bacteria within the flea vector. We use this theoretical framework to identify the environmental conditions promoting the evolution of blockage. We also show that blockage is favored at the onset of an epidemic, and that the frequencies of bacterial strains exhibiting different strategies of blockage can fluctuate in seasonal environments. This analysis quantifies the contribution of different transmission routes in plague and makes testable predictions on the adaptive nature of blockage.

Transcriptomic studies and assessment of Yersinia pestis reference genes in various conditions.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a very sensitive widespread technique considered as the gold standard to explore transcriptional variations. While a particular methodology has to be followed to provide accurate results many published studies are likely to misinterpret results due to lack of minimal quality requirements. Yersinia pestis is a highly pathogenic bacterium responsible for plague. It has been used to propose a ready-to-use and complete approach to mitigate the risk of technical biases in transcriptomic studies. The selection of suitable reference genes (RGs) among 29 candidates was performed using four different methods (GeNorm, NormFinder, BestKeeper and the Delta-Ct method). An overall comprehensive ranking revealed that 12 following candidate RGs are suitable for accurate normalization: gmk, proC, fabD, rpoD, nadB, rho, thrA, ribD, mutL, rpoB, adk and tmk. Some frequently used genes like 16S RNA had even been found as unsuitable to study Y. pestis. This methodology allowed us to demonstrate, under different temperatures and states of growth, significant transcriptional changes of six efflux pumps genes involved in physiological aspects as antimicrobial resistance or virulence. Previous transcriptomic studies done under comparable conditions had not been able to highlight these transcriptional modifications. These results highlight the importance of validating RGs prior to the normalization of transcriptional expression levels of targeted genes. This accurate methodology can be extended to any gene of interest in Y. pestis. More generally, the same workflow can be applied to identify and validate appropriate RGs in other bacteria to study transcriptional variations.

Curative Treatment of Severe Gram-Negative Bacterial Infections by a New Class of Antibiotics Targeting LpxC.

The infectious diseases caused by multidrug-resistant bacteria pose serious threats to humankind. It has been suggested that an antibiotic targeting LpxC of the lipid A biosynthetic pathway in Gram-negative bacteria is a promising strategy for curing Gram-negative bacterial infections. However, experimental proof of this concept is lacking. Here, we describe our discovery and characterization of a biphenylacetylene-based inhibitor of LpxC, an essential enzyme in the biosynthesis of the lipid A component of the outer membrane of Gram-negative bacteria. The compound LPC-069 has no known adverse effects in mice and is effective in vitro against a broad panel of Gram-negative clinical isolates, including several multiresistant and extremely drug-resistant strains involved in nosocomial infections. Furthermore, LPC-069 is curative in a murine model of one of the most severe human diseases, bubonic plague, which is caused by the Gram-negative bacterium Yersinia pestis Our results demonstrate the safety and efficacy of LpxC inhibitors as a new class of antibiotic against fatal infections caused by extremely virulent pathogens. The present findings also highlight the potential of LpxC inhibitors for clinical development as therapeutics for infections caused by multidrug-resistant bacteria.IMPORTANCE The rapid spread of antimicrobial resistance among Gram-negative bacilli highlights the urgent need for new antibiotics. Here, we describe a new class of antibiotics lacking cross-resistance with conventional antibiotics. The compounds inhibit LpxC, a key enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria, and are active in vitro against a broad panel of clinical isolates of Gram-negative bacilli involved in nosocomial and community infections. The present study also constitutes the first demonstration of the curative treatment of bubonic plague by a novel, broad-spectrum antibiotic targeting LpxC. Hence, the data highlight the therapeutic potential of LpxC inhibitors against a wide variety of Gram-negative bacterial infections, including the most severe ones caused by Y. pestis and by multidrug-resistant and extensively drug-resistant carbapenemase-producing strains.

Characterization of the protective immune response to Yersinia pseudotuberculosis infection in mice vaccinated with an LcrV-secreting strain of Lactococcus lactis.

BACKGROUND: Pseudotuberculosis is an infection caused by the bacterial enteropathogen Yersinia pseudotuberculosis and is considered to be a significant problem in veterinary medicine. We previously found that intranasal administration of a recombinant Lactococcus lactis strain that secretes the low-calcium response V (LcrV) antigen from Y. pseudotuberculosis (Ll-LcrV) confers protection against a lethal Y. pseudotuberculosis infection. Here, we aimed at characterizing the immunological basis of this LcrV-elicited protective response and at determining the duration of vaccine-induced immunity. METHODS: Splenocytes from BALB/c mice intranasally immunized with Ll-LcrV or Ll as control were immunostained then analyzed by flow cytometry. Protection against a lethal intravenous injection of Y. pseudotuberculosis was also determined (i) in immunized BALB/c mice depleted or not of CD4+, CD8+ or CD25+ cells and (ii) in naïve BALB/c mice receiving serum from immunized mice by counting the number of bacteria in liver and spleen. Lastly, survival rate of immunized BALB/c mice following a lethal intravenous injection of Y. pseudotuberculosis was followed up to 9-months. RESULTS: We found that T and B lymphocytes but not non-conventional lymphoid cells were affected by Ll-LcrV immunization. We also observed that depletion of CD4+ and CD25+ but not CD8+ cells in immunized mice eradicated protection against a lethal systemic Y. pseudotuberculosis infection, suggesting that activated CD4+ T lymphocytes are required for vaccine-induced protection. Adoptive transfer of LcrV-specific antibodies from Ll-LcrV-immunized animals significantly reduced the bacterial counts in the liver compared to non-vaccinated mice. Lastly, the protective immunity conferred by Ll-LcrV decreased slightly over time; nevertheless almost 60% of the mice survived a lethal bacterial challenge at 9months post-vaccination. CONCLUSION: Mucosal vaccination of mice with Ll-LcrV induced cell- and antibody-mediated protective immunity against Y. pseudotuberculosis infection in the mouse and the protection is long-lasting.

High susceptibility of MDR and XDR Gram-negative pathogens to biphenyl-diacetylene-based difluoromethyl-allo-threonyl-hydroxamate LpxC inhibitors.

OBJECTIVES: Inhibitors of uridine diphosphate-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC, which catalyses the first, irreversible step in lipid A biosynthesis) are a promising new class of antibiotics against Gram-negative bacteria. The objectives of the present study were to: (i) compare the antibiotic activities of three LpxC inhibitors (LPC-058, LPC-011 and LPC-087) and the reference inhibitor CHIR-090 against Gram-negative bacilli (including MDR and XDR isolates); and (ii) investigate the effect of combining these inhibitors with conventional antibiotics. METHODS: MICs were determined for 369 clinical isolates (234 Enterobacteriaceae and 135 non-fermentative Gram-negative bacilli). Time-kill assays with LPC-058 were performed on four MDR/XDR strains, including Escherichia coli producing CTX-M-15 ESBL and Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii producing KPC-2, VIM-1 and OXA-23 carbapenemases, respectively. RESULTS: LPC-058 was the most potent antibiotic and displayed the broadest spectrum of antimicrobial activity, with MIC90 values for Enterobacteriaceae, P. aeruginosa, Burkholderia cepacia and A. baumannii of 0.12, 0.5, 1 and 1 mg/L, respectively. LPC-058 was bactericidal at 1× or 2× MIC against CTX-M-15, KPC-2 and VIM-1 carbapenemase-producing strains and bacteriostatic at ≤4× MIC against OXA-23 carbapenemase-producing A. baumannii. Combinations of LPC-058 with ß-lactams, amikacin and ciprofloxacin were synergistic against these strains, albeit in a species-dependent manner. LPC-058's high efficacy was attributed to the presence of the difluoromethyl-allo-threonyl head group and a linear biphenyl-diacetylene tail group. CONCLUSIONS: These in vitro data highlight the therapeutic potential of the new LpxC inhibitor LPC-058 against MDR/XDR strains and set the stage for subsequent in vivo studies.

Drug design from the cryptic inhibitor envelope.

Conformational dynamics plays an important role in enzyme catalysis, allosteric regulation of protein functions and assembly of macromolecular complexes. Despite these well-established roles, such information has yet to be exploited for drug design. Here we show by nuclear magnetic resonance spectroscopy that inhibitors of LpxC--an essential enzyme of the lipid A biosynthetic pathway in Gram-negative bacteria and a validated novel antibiotic target--access alternative, minor population states in solution in addition to the ligand conformation observed in crystal structures. These conformations collectively delineate an inhibitor envelope that is invisible to crystallography, but is dynamically accessible by small molecules in solution. Drug design exploiting such a hidden inhibitor envelope has led to the development of potent antibiotics with inhibition constants in the single-digit picomolar range. The principle of the cryptic inhibitor envelope approach may be broadly applicable to other lead optimization campaigns to yield improved therapeutics.

Evaluation of the Role of the opgGH Operon in Yersinia pseudotuberculosis and Its Deletion during the Emergence of Yersinia pestis.

The opgGH operon encodes glucosyltransferases that synthesize osmoregulated periplasmic glucans (OPGs) from UDP-glucose, using acyl carrier protein (ACP) as a cofactor. OPGs are required for motility, biofilm formation, and virulence in various bacteria. OpgH also sequesters FtsZ in order to regulate cell size according to nutrient availability. Yersinia pestis (the agent of flea-borne plague) lost the opgGH operon during its emergence from the enteropathogen Yersinia pseudotuberculosis. When expressed in OPG-negative strains of Escherichia coli and Dickeya dadantii, opgGH from Y. pseudotuberculosis restored OPGs synthesis, motility, and virulence. However, Y. pseudotuberculosis did not produce OPGs (i) under various growth conditions or (ii) when overexpressing its opgGH operon, its galUF operon (governing UDP-glucose), or the opgGH operon or Acp from E. coli. A ΔopgGH Y. pseudotuberculosis strain showed normal motility, biofilm formation, resistance to polymyxin and macrophages, and virulence but was smaller. Consistently, Y. pestis was smaller than Y. pseudotuberculosis when cultured at ≥ 37°C, except when the plague bacillus expressed opgGH. Y. pestis expressing opgGH grew normally in serum and within macrophages and was fully virulent in mice, suggesting that small cell size was not advantageous in the mammalian host. Lastly, Y. pestis expressing opgGH was able to infect Xenopsylla cheopis fleas normally. Our results suggest an evolutionary scenario whereby an ancestral Yersinia strain lost a factor required for OPG biosynthesis but kept opgGH (to regulate cell size). The opgGH operon was presumably then lost because OpgH-dependent cell size control became unnecessary.