Humoral and Cellular Immune Responses to Yersinia pestis Infection in Long-Term Recovered Plague Patients

Plague is one of the most dangerous diseases and is caused by Yersinia pestis. Effective vaccine development requires understanding of immune protective mechanisms against the bacterium in humans. In this study, the humoral and memory cellular immune responses in plague patients (n = 65) recovered from Y. pestis infection during the past 16 years were investigated using a protein microarray and an enzyme-linked immunosorbent spot assay (ELISpot). The seroprevalence to the F1 antigen in all recovered patients is 78.5%. In patients infected more than a decade ago, the antibody-positive rate still remains 69.5%. There is no difference in the antibody presence between gender, age, and infected years, but it seems to be associated with the F1 antibody titers during infection (r = 0.821; P < 0.05). Except F1 antibody, the antibodies against LcrV and YopD were detected in most of the patients, suggesting they could be the potential diagnostic markers for detecting the infection of F1-negative strains. Regarding cellular immunity, the cell number producing gamma interferon (IFN-γ), stimulated by F1 and LcrV, respectively, in vitro to the peripheral blood mononuclear cells of 7 plague patients and 4 negative controls, showed no significant difference, indicating F1 and LcrV are not dominant T cell antigens against plague for a longer time in humans. Our findings have direct implications for the future design and development of effective vaccines against Y. pestis infection and the development of new target-based diagnostics.     

Deletion of Braun Lipoprotein and Plasminogen-Activating Protease-Encoding Genes Attenuates Yersinia pestis in Mouse Models of Bubonic and Pneumonic Plague

Currently, there is no FDA-approved vaccine against Yersinia pestis, the causative agent of bubonic and pneumonic plague. Since both humoral immunity and cell-mediated immunity are essential in providing the host with protection against plague, we developed a live-attenuated vaccine strain by deleting the Braun lipoprotein (lpp) and plasminogen-activating protease (pla) genes from Y. pestis CO92. The Δlpp Δpla double isogenic mutant was highly attenuated in evoking both bubonic and pneumonic plague in a mouse model. Further, animals immunized with the mutant by either the intranasal or the subcutaneous route were significantly protected from developing subsequent pneumonic plague. In mice, the mutant poorly disseminated to peripheral organs and the production of proinflammatory cytokines concurrently decreased. Histopathologically, reduced damage to the lungs and livers of mice infected with the Δlpp Δpla double mutant compared to the level of damage in wild-type (WT) CO92-challenged animals was observed. The Δlpp Δpla mutant-immunized mice elicited a humoral immune response to the WT bacterium, as well as to CO92-specific antigens. Moreover, T cells from mutant-immunized animals exhibited significantly higher proliferative responses, when stimulated ex vivo with heat-killed WT CO92 antigens, than mice immunized with the same sublethal dose of WT CO92. Likewise, T cells from the mutant-immunized mice produced more gamma interferon (IFN-γ) and interleukin-4. These animals had an increasing number of tumor necrosis factor alpha (TNF-α)-producing CD4⁺ and CD8⁺ T cells than WT CO92-infected mice. These data emphasize the role of TNF-α and IFN-γ in protecting mice against pneumonic plague. Overall, our studies provide evidence that deletion of the lpp and pla genes acts synergistically in protecting animals against pneumonic plague, and we have demonstrated an immunological basis for this protection.     

Kinetics of Innate Immune Response to Yersinia pestis after Intradermal Infection in a Mouse Model

A hallmark of Yersinia pestis infection is a delayed inflammatory response early in infection. In this study, we use an intradermal model of infection to study early innate immune cell recruitment. Mice were injected intradermally in the ear with wild-type (WT) or attenuated Y. pestis lacking the pYV virulence plasmid (pYV⁻). The inflammatory responses in ear and draining lymph node samples were evaluated by flow cytometry and immunohistochemistry. As measured by flow cytometry, total neutrophil and macrophage recruitment to the ear in WT-infected mice did not differ from phosphate-buffered saline (PBS) controls or mice infected with pYV⁻, except for a transient increase in macrophages at 6 h compared to the PBS control. Limited inflammation was apparent even in animals with high bacterial loads (10⁵ to 10⁶ CFU). In addition, activation of inflammatory cells was significantly reduced in WT-infected mice as measured by CD11b and major histocompatibility complex class II (MHC-II) expression. When mice infected with WT were injected 12 h later at the same intradermal site with purified LPS, Y. pestis did not prevent recruitment of neutrophils. However, significant reduction in neutrophil activation remained compared to that of PBS and pYV⁻ controls. Immunohistochemistry revealed qualitative differences in neutrophil recruitment to the skin and draining lymph node, with WT-infected mice producing a diffuse inflammatory response. In contrast, focal sites of neutrophil recruitment were sustained through 48 h postinfection in pYV⁻-infected mice. Thus, an important feature of Y. pestis infection is reduced activation and organization of inflammatory cells that is at least partially dependent on the pYV virulence plasmid.     

The N Terminus of Type III Secretion Needle Protein YscF from Yersinia pestis Functions To Modulate Innate Immune Responses

The type III secretion system is employed by many pathogens, including the genera Yersinia, Shigella, Pseudomonas, and Salmonella, to deliver effector proteins into eukaryotic cells. The injectisome needle is formed by the polymerization of a single protein, e.g., YscF (Yersinia pestis), PscF (Pseudomonas aeruginosa), PrgI (Salmonella enterica SPI-1), SsaG (Salmonella enterica SPI-2), or MxiH (Shigella flexneri). In this study, we demonstrated that the N termini of some needle proteins, particularly the N terminus of YscF from Yersinia pestis, influences host immune responses. The N termini of several needle proteins were truncated and tested for the ability to induce inflammatory responses in a human monocytic cell line (THP-1 cells). Truncated needle proteins induced proinflammatory cytokines to different magnitudes than the corresponding wild-type proteins, except SsaG. Notably, N-terminally truncated YscF induced significantly higher activation of NF-κB and/or AP-1 and higher induction of proinflammatory cytokines, suggesting that a function of the N terminus of YscF is interference with host sensing of YscF, consistent with Y. pestis pathogenesis. To directly test the ability of the N terminus of YscF to suppress cytokine induction, a YscF-SsaG chimera with 15 N-terminal amino acids from YscF added to SsaG was constructed. The chimeric YscF-SsaG induced lower levels of cytokines than wild-type SsaG. However, the addition of 15 random amino acids to SsaG had no effect on NF-κB/AP-1 activation. These results suggest that the N terminus of YscF can function to decrease cytokine induction, perhaps contributing to a favorable immune environment leading to survival of Y. pestis within the eukaryotic host.     

Lymphocyte Circadian Clocks Control Lymph Node Trafficking and Adaptive Immune Responses

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Gr1+ Cells Control Growth of YopM-Negative Yersinia pestis during Systemic Plague

YopM, a protein toxin of Yersinia pestis, is necessary for virulence in a mouse model of systemic plague. We previously reported YopM-dependent natural killer (NK) cell depletion from blood and spleen samples of infected mice. However, in this study we found that infection with Y. pestis KIM5 (YopM⁺) caused depletion of NK cells in the spleen, but not in the liver, and antibody-mediated ablation of NK cells had no effect on bacterial growth. There was no YopM-associated effect on the percentage of dendritic cells (DCs) or polymorphonuclear leukocytes (PMNs) in the early stage of infection; however, there was a YopM-associated effect on PMN integrity and on the influx of monocytes into the spleen. Ablation of Gr1⁺ cells caused loss of the growth defect of YopM⁻ Y. pestis in both the liver and spleen. In contrast, ablation of macrophages/DCs inhibited growth of both parent and mutant bacteria, accompanied by significantly fewer lesion sites in the liver. These results point toward PMNs and inflammatory monocytes as major cell types that control growth of YopM⁻ Y. pestis. Infection with fully virulent Y. pestis CO92 and a YopM⁻ derivative by intradermal and intranasal routes showed that the absence of YopM significantly increased the 50% lethal dose only in the intradermal model, suggesting a role for YopM in bubonic plague, in which acute inflammation occurs soon after infection.Yersinia pestis, the infective agent of bubonic and pneumonic plague, has caused widespread loss of human life during recurrent pandemics. Y. pestis still infects rodent populations in large geographic zones where Y. pestis is endemic, and there are cases of human plague reported annually (15, 19, 56). Y. pestis and the closely related food-borne pathogens Yersinia pseudotuberculosis and Yersinia enterocolitica share a 70-kb plasmid carrying genes that encode a major set of proteins involved in pathogenic properties that compromise the host immune system (60). These include a type 3 secretion system (T3SS) that at mammalian body temperature delivers a set of six Yersinia outer protein (Yop) effector proteins into host cells once the bacteria contact host target cells. Enzymatic and cell biological mechanisms of five of the Yops, YopH, YopE, YopT, YpkA/YopO, and YopJ, have been elucidated. YopJ interferes with signal transduction through acetyltransferase activity but is not required for virulence in either a mouse model of systemic plague (57) or mouse and rat models of bubonic plague (28, 65). In tissue culture infection models, YopH, YopE, YopT, and YopO have been shown to antagonize focal complex formation and activity of Rho family GTPases and synergistically inhibit phagocytosis by mammalian cells. YopH and YopE have been shown to be crucial for lethality in a mouse model of systemic plague (intravenous [i.v.] infection), and a ΔyopH strain is attenuated for both bubonic and pneumonic plague (9). In addition, Y. pestis virulence proteins, such as the surface fibrils F1 and PsaA, have antiphagocytic effects and also have been found to contribute to virulence in systemic plague (7, 31). Accordingly, Y. pestis is believed to exist predominantly in an extracellular location in vivo, although initially the bacteria might invade resting tissue macrophages (Mφs) and dendritic cells (DCs), based on assays of mouse spleens in the systemic phase of bubonic plague (33). The intracellular versus extracellular locations of Y. pestis during the peripheral phases of plague on skin or in the lung have not yet been studied.It is believed that tissue Mφs, DCs, and polymorphonuclear leukocytes (PMNs) are early target cells for Yop delivery in vivo, because these cells are present before or soon after infection begins and function to initiate the innate defenses that are undermined by Yops. Consistent with this hypothesis, Y. pestis has been found in association with alveolar Mφs early during lung infection of mice (6) and likewise in association with Mφs, DCs, and PMNs in the spleens of mice infected i.v., and YopM can be injected into these cells (34). However, it is becoming clear that spleens and lungs present distinctly different inflammatory environments when infected by Y. pestis, with PMNs migrating rapidly into spleens infected by the i.v. route but not appearing in lungs until 36 h after intranasal (i.n.) infection (27, 58). Accordingly, some virulence properties required for lethality of systemic plague are not required in pneumonic plague. Examples are the capsular fibril F1 and the antiphagocytic adhesin PsaA (7, 11, 12). There is no information yet on the target cells or relative importance of Yops other than YopH in pneumonic or bubonic plague.The sixth effector Yop, YopM, is essential for virulence in the mouse model of systemic plague: in C57BL/6 mice, a YopM⁻ strain of Y. pestis KIM5 is reduced in lethality by at least 4 orders of magnitude (29). However, the function of YopM has not been defined. YopM is a 46.2-kDa acidic protein made up almost entirely of 15 repeats of a 19-residue leucine-rich repeat motif (30). The YopM monomer is horseshoe shaped and has the potential to form tetramers in which the monomers stack together to form a hollow cylinder; however, the form that YopM assumes within the mammalian cell is not known (16). After delivery to the host cell cytoplasm, YopM localizes to the nucleus in a process that is facilitated by vesicular trafficking (53). YopM was reported to form a complex with the serine/threonine kinases PRK2 (protein kinase C-related kinase 2) and RSK1 (90-kDa ribosomal S6 kinase) in HEK293 cells infected with Y. pseudotuberculosis (36), leading to activation of both kinases. However, the biological significance of this complex is not known. There is no visible effect of delivery of YopM into cultured cells, and microarray analysis of Mφ-like cell lines infected with Y. enterocolitica having or lacking YopM also has not yielded any clue to YopM''s mechanism of action (21, 50).Because these and other in vitro approaches to defining the pathogenic mechanism of YopM have not been fruitful, we have begun to characterize YopM''s effects in vivo. Previously we found that YopM was still required for lethality in i.v. infected SCID mice, showing that YopM''s virulence mechanism does not require B or T cells and indicating that early in systemic plague, YopM''s main function is to counteract a component of innate immunity (25). A striking YopM-specific effect during systemic plague in wild-type C57BL/6 mice was the depletion of natural killer (NK) cells from the spleen and a reduction of NK cell numbers in blood, suggesting that YopM might cause a loss of the NK cell compartment during systemic plague. Correlated with this effect, there was a YopM-associated loss of mRNA for gamma interferon (IFN-γ) by NK cells in infected spleens and diminished expression of mRNAs in splenic Mφs for cytokines that are required for viability and activation of NK cells (interleukin 15 [IL-15], IL-18, and IL-12). These findings supported the hypothesis that YopM may function to inhibit IFN-mediated activation of Mφs through the depletion of NK cells (25).In this study, we tested the hypothesis that NK cells are critical for controlling Y. pestis pathogenesis early in plague and that NK cells are the primary target of YopM. We characterized infection dynamics and leukocyte populations in both the liver and spleen during systemic plague in mice ablated for Mφs/DCs or for Gr1⁺ cells. The data point to PMNs and inflammatory monocytes as critical cells affected by YopM and to Mφs/DCs as an important early reservoir for bacterial growth. Consistent with a role in undermining acute inflammation, YopM was found to be important for the lethality of bubonic plague, but not pneumonic plague.     

Dual-Function Antibodies to Yersinia pestis LcrV Required for Pulmonary Clearance of Plague

Yersinia pestis causes pneumonic plague, a necrotic pneumonia that rapidly progresses to death without early treatment. Antibodies to the protective antigen LcrV are thought to neutralize its essential function in the type III secretion system (TTSS) and by themselves are capable of inducing immunity to plague in mouse models. To develop multivalent LcrV antibodies as a therapeutic treatment option, we screened for monoclonal antibodies (MAbs) to LcrV that could prevent its function in the TTSS. Although we were able to identify single and combination MAbs that provided the high-level inhibition of the TTSS, these did not promote phagocytosis in vitro and were only weakly protective in a mouse pneumonic plague model. Only one MAb, BA5, was able to protect mice from pneumonic plague. In vitro, MAb BA5 blocked the TTSS with efficiency equal to or even less than that of other MAbs as single agents or as combinations, but its activity led to increased phagocytic uptake. Polyclonal anti-LcrV was superior to BA5 in promoting phagocytosis and also was more efficient in protecting mice from pneumonic plague. Taken together, the data support a hypothesis whereby the pulmonary clearance of Y. pestis by antibodies requires both the neutralization of the TTSS and the simultaneous stimulation of innate signaling pathways used by phagocytic cells to destroy pathogens.Yersinia pestis, the etiologic agent of bubonic, pneumonic, and septicemic plague, has been responsible for more human death than any other bacterial pathogen (42). Fortunately, naturally occurring cases of plague in humans now are uncommon, largely due to advances in basic sanitation and public awareness of infectious disease (32). Nevertheless, the disease remains endemic in many areas of the world, and periodic human bubonic and, to a lesser extent, pneumonic plague cases appear each year. Yersinia pestis is believed to have evolved recently from Yersinia pseudotuberculosis, acquiring flea transmission and respiratory invasion properties through mobile genetic elements (1, 9). The flea transmission cycle provides an opportunity for further evolution, because the bacteria reside in the nonsterile environment of the flea gut, where the formation of a biofilm provides an opportunity for horizontal gene exchange with other microbes (30). Multidrug-resistant Y. pestis isolates have been recovered from human plague patients, suggesting that the bacteria do indeed continue to evolve mechanisms of survival in the mammalian host (22, 25, 54). For these reasons, as well as for its potential use as a biological weapon, Y. pestis continues to be a significant public health concern and is a priority pathogen for the development of new vaccines and alternative therapeutics (32, 43).There currently are no plague vaccines that are licensed for human use in the United States. The licensing of current candidates is likely to fall under the U.S. Food and Drug Administration''s Animal Rule for the demonstration of efficacy and potency due to a lack of naturally occurring human plague cases (19). Thus, efficacy trials and the evaluation of vaccine potency in humans will be dependent on our ability to understand the molecular mechanism of protection. Current subunit vaccine candidates are formulated from two protective antigens, Fraction 1 (F1) and LcrV, which are undergoing extensive testing to satisfy the Animal Rule requirements (2, 5, 13, 26, 55, 57-59). Both antigens elicit a neutralizing antibody response that can be translated to passive antibody or even gene therapies (2, 4, 13, 28, 37, 48). These protective antibodies act directly on the bacteria and alter its interactions with innate immune cells such that the host clears the infection. T-cell responses also are believed to play an important role in host defense against Yersinia pestis (40, 41).CaF1, or F1, is an abundant cell surface antigen of the type I pilin family that forms a capsule-like structure on Y. pestis at 37°C (8). Although F1 appears to be antiphagocytic, it is not essential for virulence and thus would not contribute to immunity against Y. pestis mutant caF1 (18, 21). In contrast, LcrV is essential for all forms of plague due to its role in the type III secretion system (TTSS) (12, 45, 47). LcrV is positioned on the surface of bacteria at 37°C, where it mediates the translocation of anti-host factors, collectively known as Yersinia outer proteins (Yops), whose antiphagocytic, cytolytic, and proapoptotic activities allow Yersinia to avoid being killed by the host''s immune system (38, 46). Polyclonal antibodies to recombinant LcrV (α-LcrV) can bind to this needle tip and lead to the inhibition of the TTSS and the phagocytosis of the bacteria (14, 24, 53). However, it remains controversial whether the direct inhibition of the TTSS by α-LcrV leads to phagocytosis or if the direct promotion of phagocytosis leads to the inhibition of the TTSS because it cannot function intracellularly (59, 60). Three monoclonal antibodies (MAbs) have been independently cloned that can protect mice from bubonic and pneumonic plague (2, 27, 48). Although it is unclear whether each of these targets the same epitope, deletion studies of LcrV antigen suggest multiple protective epitopes exist (13, 39, 44, 51).We were interested in developing antibody therapeutics and maximizing the potency of anti-LcrV therapy. In this work, we investigated the mechanism of protection from pneumonic plague to determine if the multivalent occupancy of antibody to LcrV improved protection. We found that antibodies that promoted phagocytosis directly were more potent at neutralizing pneumonic plague, although the inhibition of the TTSS alone led to partial protection. Only a single LcrV epitope led to antibodies that by themselves promoted uptake, while the multivalent occupation of antigen with MAbs did not increase either phagocytosis or protection. These data provide new insight into the mechanism of LcrV and support the use of assays that measure the phagocytic uptake of Y. pestis as correlates of immunity for the evaluation of plague vaccines.     

Plague in Algeria: about five strains of Yersinia pestis isolated during the outbreak of June 2003

In this study, we isolated and identified five strains of Yersinia pestis during an epidemic occurred in west of Algeria in June 2003. The bacteriological identification was confirmed by bacteriophage susceptibility. All these strains belonged to the biovar Orientalis (they did not ferment glycerol but did reduce nitrate to nitrite) which caused the current pandemic. The in vitro activities of antimicrobial agents used to treat plague and recommended for prophylaxis, showed that they are active against all strains. The comparison of these strains by plasmid profile analyse demonstrated that all isolates had three plasmids: 110, 70 and 9.5 kb, which are present in Y. pestis strains.     

Three Yersinia pestis Adhesins Facilitate Yop Delivery to Eukaryotic Cells and Contribute to Plague Virulence

To establish a successful infection, Yersinia pestis requires the delivery of cytotoxic Yops to host cells. Yops inhibit phagocytosis, block cytokine responses, and induce apoptosis of macrophages. The Y. pestis adhesin Ail facilitates Yop translocation and is required for full virulence in mice. To determine the contributions of other adhesins to Yop delivery, we deleted five known adhesins of Y. pestis. In addition to Ail, plasminogen activator (Pla) and pH 6 antigen (Psa) could mediate Yop translocation to host cells. The contribution of each adhesin to binding and Yop delivery was dependent upon the growth conditions. When cells were pregrown at 28°C and pH 7, the order of importance for adhesins in cell binding and cytotoxicity was Ail > Pla > Psa. Y. pestis grown at 37°C and pH 7 had equal contributions from Ail and Pla but an undetectable role for Psa. At 37°C and pH 6, both Ail and Psa contributed to binding and Yop delivery, while Pla contributed minimally. Pla-mediated Yop translocation was independent of protease activity. Of the three single mutants, the Δail mutant was the most defective in mouse virulence. The expression level of ail was also the highest of the three adhesins in infected mouse tissues. Compared to an ail mutant, additional deletion of psaA (encoding Psa) led to a 130,000-fold increase in the 50% lethal dose for mice relative to that of the KIM5 parental strain. Our results indicate that in addition to Ail, Pla and Psa can serve as environmentally specific adhesins to facilitate Yop secretion, a critical virulence function of Y. pestis.The causative agent of plague is the Gram-negative bacterium Yersinia pestis (54, 77). Plague is one of the most deadly infectious diseases and has decimated civilizations repeatedly throughout history (10, 54). Plague still remains a public health concern, and due to the increasing number of cases worldwide, plague is classified as a reemerging infectious disease (68). Identification of therapies or effective vaccines would provide protection against plague as a potential bioterrorism threat.There are three clinical forms of plague in humans: bubonic, pneumonic, and septicemic (54). Bubonic plague is the most common form and usually occurs following a fleabite. In bubonic plague, the organism initially spreads to the regional lymph nodes, where it replicates primarily extracellularly, and then eventually enters the bloodstream. If untreated, bubonic plague is fatal in 40 to 60% of cases (54). Pneumonic plague is the least common form, but it progresses rapidly and is the most fatal form of the disease. Pneumonic plague may occur as a complication of bubonic or septicemic plague (secondary pneumonic plague) or by inhalation of infectious droplets spread by the cough or sneeze of a person with pneumonic plague (primary pneumonic plague). If treatment is not initiated within the first 24 h after symptoms appear, it is likely to be fatal within 48 h (18, 49). Septicemic plague can occur if Y. pestis gains direct access to the bloodstream via open wounds or fleabites (primary septicemic plague) (64) or as a result of spread from the lymphatic system to the bloodstream during advanced stages of bubonic plague (54). Y. pestis can also spread to the bloodstream and blood-filtering organs during late stages of pneumonic plague (39).In order for Y. pestis to cause disease, it must harbor the 70-kb pCD1 virulence plasmid, which encodes the Ysc type III secretion system (T3SS) and the Yop effector proteins (13, 69, 70). Yops inhibit phagocytosis by disrupting the actin cytoskeleton, diminish proinflammatory cytokine responses, and induce apoptosis of macrophages (13, 30, 50, 51, 54, 60, 62). In order for Yops to be delivered efficiently to host cells, adhesins must provide a docking function to facilitate T3S (8, 22, 59).Two adhesins shown to be important for Yop delivery in the related Yersinia species Y. enterocolitica and Y. pseudotuberculosis are YadA and invasin (Inv) (8, 47, 59). Y. pestis lacks both of these adhesins due to inactivation of inv by an IS1541 element and of yadA by a frameshift mutation (17, 52, 61, 65). Thus, we focused our studies on the Y. pestis adhesins described below.We recently identified Ail, an adhesin of Y. pestis that binds host cell fibronectin (73) and plays an important role in delivery of Yops to both phagocytic and nonphagocytic cells in vitro (22). In addition to having a defect in Yop delivery in vitro, a Y. pestis KIM5 Δail mutant has a >3,000-fold increased 50% lethal dose (LD₅₀) for mice in a septicemic plague model (22). However, the virulence defect of a KIM5 Δail mutant is not as severe as that of a KIM5 strain lacking the virulence plasmid (>10⁶-fold increase in LD₅₀) (22, 69). Thus, we set out to determine if there were other Y. pestis adhesins capable of facilitating Yop delivery.Four other adhesins of Y. pestis have been described. Plasminogen activator (Pla) is an adhesin and a protease. It can mediate binding to extracellular matrix proteins (32, 37, 43) and direct invasion of tissue culture cells (14, 36). Pla may also interact with the host cell receptor DEC-205 on macrophages and dendritic cells (80). Pla is known to be required for dissemination of a bubonic plague infection from the site of inoculation, presumably due to cleavage of fibrin clots by plasmin after Pla-mediated activation of plasminogen (27, 66). Its protease activity is also required for the development of fulminant pneumonic plague (39).pH 6 antigen (Psa) is a multifunctional surface structure that is induced at 37°C under low-pH conditions (4, 57, 75) and is controlled by the regulatory protein RovA (11). Psa can bind to β1-linked galactosyl residues in glycosphingolipids (53) and to phosphatidylcholine (26) on the surfaces of host cells. Due to its ability to bind surface receptors, Psa functions as an efficient adhesin (5, 24, 26, 41, 76). Psa-mediated adhesion also prevents phagocytosis (29). Finally, Psa can bind the Fc portion of human IgG (79) and interact with plasma low-density lipoproteins (LDLs) (44).The two remaining Y. pestis adhesins are the putative autotransporter YapC (23) and the pilus associated with the chaperone usher locus y0561 to y0563 (24). Neither locus is well expressed under laboratory conditions (23, 24; S. Felek and E. S. Krukonis, unpublished data), but when expressed in Escherichia coli, they can confer an increase in E. coli adhesion to several cultured cell lines (23, 24).Here we demonstrate that Ail, Pla, and Psa can mediate Yop delivery in vitro when expressed in a KIM5 mutant lacking all five adhesins (KIM5 Δ5), whereas overexpression of YapC or y0561 to y0563 does not allow for Yop delivery from the KIM5 Δ5 strain. Furthermore, we observed cumulative defects in virulence when each of three genes, ail, pla, and psaA, was deleted and tested in a mouse model. Thus, we have shown that all three adhesins can contribute to Yop delivery in vitro and to virulence in vivo.     

GSL-Enriched Membrane Microdomains in Innate Immune Responses

Many pathogens target glycosphingolipids (GSLs), which, together with cholesterol, GPI-anchored proteins, and various signaling molecules, cluster on host cell membranes to form GSL-enriched membrane microdomains (lipid rafts). These GSL-enriched membrane microdomains may therefore be involved in host–pathogen interactions. Innate immune responses are triggered by the association of pathogens with phagocytes, such as neutrophils, macrophages and dendritic cells. Phagocytes express a diverse array of pattern-recognition receptors (PRRs), which sense invading microorganisms and trigger pathogen-specific signaling. PRRs can recognize highly conserved pathogen-associated molecular patterns expressed on microorganisms. The GSL lactosylceramide (LacCer, CDw17), which binds to various microorganisms, including Candida albicans, is expressed predominantly on the plasma membranes of human mature neutrophils and forms membrane microdomains together with the Src family tyrosine kinase Lyn. These LacCer-enriched membrane microdomains can mediate superoxide generation, migration, and phagocytosis, indicating that LacCer functions as a PRR in innate immunity. Moreover, the interactions of GSL-enriched membrane microdomains with membrane proteins, such as growth factor receptors, are important in mediating the physiological properties of these proteins. Similarly, we recently found that interactions between LacCer-enriched membrane microdomains and CD11b/CD18 (Mac-1, CR3, or αMβ2-integrin) are significant for neutrophil phagocytosis of non-opsonized microorganisms. This review describes the functional role of LacCer-enriched membrane microdomains and their interactions with CD11b/CD18.     

Immune response to Yersinia outer proteins and other Yersinia pestis antigens after experimental plague infection in mice

There is limited information concerning the nature and extent of the immune response to the virulence determinants of Yersinia pestis during the course of plague infection. In this study, we evaluated the humoral immune response of mice that survived lethal Y. pestis aerosol challenge after antibiotic treatment. Such a model may replicate the clinical situation in humans and indicate which virulence determinants are expressed in vivo. Immunoglobulin G enzyme-linked immunosorbent assay and immunoblotting were performed by using purified, recombinant antigens including F1, V antigen, YpkA, YopH, YopM, YopB, YopD, YopN, YopE, YopK, plasminogen activator protease (Pla), and pH 6 antigen as well as purified lipopolysaccharide. The major antigens recognized by murine convalescent sera were F1, V antigen, YopH, YopM, YopD, and Pla. Early treatment with antibiotics tended to reduce the immune response and differences between antibiotic treatment regimens were noted. These results may indicate that only some virulence factors are expressed and/or immunogenic during infection. This information may prove useful for selecting potential vaccine candidates and for developing improved serologic diagnostic assays.     

Immune responses of two Mastomys sibling species to Yersinia pestis.

This study assessed the in vitro cell-mediated immune responses of Mastomys natalensis, with a diploid chromosome number of 2n = 32, and Mastomys coucha, with a diploid chromosome number of 2n = 36, to Yersinia pestis. Splenic mononuclear (MN) cells of uninfected M. natalensis proliferated in response to crude fraction 1 of Y. pestis and two subfractions derived from fraction 1 in vitro. Proliferation was dose dependent and followed the time kinetics of other well-known mitogens. Further characterization of the two fractions revealed similar protein profiles in sodium dodecyl sulfide-polyacrylamide gel electrophoresis and indicated a heat-stable protein of 25 kDa responsible for the mitogenic activity. No such response was observed with MN cells from M. coucha. The unresponsiveness of M. coucha-derived MN cells appears to be related to an inability to respond to Y. pestis organisms. The results may help explain the relative resistance and susceptibility of M. natalensis and M. coucha to Y. pestis infection.     

Editorial: CpG Motifs to Modulate Innate and Adaptive Immune Responses

The vertebrate adaptive and innate immune systems have evolved to protect the host from pathogen infections. To achieve this mission, the innate immune system developed particular receptors, termed “pattern recognition receptors” (PRRs). These PRRs selectively bind certain types of structures expressed by pathogens but in principal absent in vertebrates. One of the best understood receptors is the Toll-like receptor (TLR) 9 that recognizes CpG sequence motifs in bacterial and viral DNA. Different classes of short synthetic phosphorothioate-stabilized CpG oligodeoxynucleotides were developed and are currently in human clinical trials in the fields of infectious disease, cancer, and asthma/allergy.     

The Lymph Node Revisited: Development,Morphology, Functioning,and Role in Triggering Primary Immune Responses

Scenarios have been proposed to explain how lymphoid components of a lymph node favor the encounter of a drained antigen with a circulating competent naïve lymphocyte to trigger a primary immune response. However, these scenarios rest on incorrect concepts about the organ. This situation resulted from a loss of interest for studies on in vivo lymphoid organs due to a widespread switch, decades ago, to work on suspended lymphoid cells. However, an in vivo holistic study of the organ continued in our laboratory. The present review synthesizes resulting knowledge on lymph node morphology and global functioning. We show that the opening of an afferent lymphatic vessel into the subcapsular sinus is the focal point from which the related portion of a lymph node—a node compartment—is developed. As to the formation of a compartment's lymphoid components, it is neonatally orchestrated by the dichotomic nature and distribution of antigens in this subcapsular sinus, which determines a dichotomic recruitment of circulating cells and the compartment's architectural complexity. The transport process of an antigen from a given tissue territory into restricted sites of the draining compartment further defines its local morphological features and activities, while providing the possibility to reduce the wandering of a short‐lived naïve cell through innumerable target‐devoid sites. We also explain that the nodal lymphoid components are not implicated in the triggering of primary responses, but are rather products of such responses. Scenarios for the triggering of primary responses, consistent with real node morphology and functioning, are proposed. Anat Rec, 293:320–337, 2010. © 2010 Wiley‐Liss, Inc.