Multispecies Epidemiologic Surveillance Study after an Outbreak of Yersiniosis at an African Green Monkey Research Facility

After an outbreak of Yersinia enterocolitica at a NHP research facility, we performed a multispecies investigation of the prevalence of Yersinia spp. in various mammals that resided or foraged on the grounds of the facility, to better understand the epizootiology of yersiniosis. Blood samples and fecal and rectal swabs were obtained from 105 captive African green monkeys (AGM), 12 feral cats, 2 dogs, 20 mice, 12 rats, and 3 mongooses. Total DNA extracted from swab suspensions served as template for the detection of Y. enterocolitica DNA by real-time PCR. Neither Y. enterocolitica organisms nor their DNA were detected from any of these samples. However, Western blotting revealed the presence of Yersinia antibodies in plasma. The AGM samples revealed a seroprevalence of 91% for Yersinia spp. and of 61% for Y. enterocolitica specifically. The AGM that were housed in cages where at least one fatality occurred during the outbreak (clinical group) had similar seroprevalence to that of AGM housed in unaffected cages (nonclinical group). However, the nonclinical group was older than the clinical group. In addition, 25%, 100%, 33%, 10%, and 10% of the sampled local cats, dogs, mongooses, rats, and mice, respectively, were seropositive. The high seroprevalence after this outbreak suggests that Y. enterocolitica was transmitted effectively through the captive AGM population and that age was an important risk factor for disease. Knowledge regarding local environmental sources of Y. enterocolitica and the possible role of wildlife in the maintenance of yersiniosis is necessary to prevent and manage this disease.Abbreviations: AGM, African green monkeyYersinia enterocolitica is a zoonotic, gram-negative member of the family Enterobacteriaceae and the causative agent of mesenteric lymphadenitis, terminal ileitis, acute gastroenteritis, and septicemia in domestic animals, wildlife, and primates. The bacterium has a very broad host range and has been detected in more than 110 species of animals worldwide, including mammals, birds, and reptiles.^3,4,19 People of all ages can become infected with pathogenic strains of Y. enterocolitica. Clinical illness is more frequent in children and young adults, with asymptomatic infection being more common in adults.²³ Latent infection by Y. enterocolitica occurs in free-living wild rodents, which excrete the organism in their feces.^6,14 Contaminated food and water are common vehicles for the transmission of this pathogen.^5,10Y. enterocolita presents high antigenic variability. There are approximately 34 O-antigen and 20 H-antigen serogroups.¹² In primates, serotypes O3, O5/27, and O9 have relatively low pathogenicity, mainly causing diarrhea, but serotype O8 is highly pathogenic and can cause septicemia.^11,24 NHP appear to be quite susceptible to infection with Y. enterocolitica, and many fatal cases of yersiniosis have been reported worldwide.^3,4,16,24,26In 2012, Y. enterocolitica was identified as the causative agent of an outbreak in captive African green monkeys (AGM; Chlorocebus sabaeus) on the island of St Kitts, West Indies, where approximately 4% of AGM in a local research facility died. Affected AGM presented with mucohemorrhagic diarrhea, marked dehydration, lethargy, and depression, often followed by death. Samples of the spleen, liver and lungs of affected monkeys yielded 15 bacterial isolates, all of which were identified as Y. enterocolitica by biochemical analysis and sequence comparison of the 16S rRNA gene. Phenotypic and genotypic analysis of the recovered isolates revealed homogeneity among the recovered bacteria, and all isolates gave a random amplified polymorphic DNA pattern resembling that of genotype D in serotypes O:7,8.²⁷The objectives of the current study were to: 1) describe the prevalence of yersiniosis in AGM, dogs (Canis lupus familaris), cats (Felis catus), and peridomestic wildlife including small Asian mongooses (Herpestes javanicus), mice (Mus spp.) and rats (Rattus norvegicus) that resided or foraged within the perimeter of the NHP research institute where the outbreak of yersiniosis occurred; 2) identify potential reservoirs of infection; and 3) better understand the epizootiology of this pathogen in the Caribbean. This information may be helpful for developing reliable and sensitive diagnostic methods and can serve as a baseline for developing effective biosecurity protocols and prophylactic measures, including vaccination and the use of probiotics.     

Epidemiology of Invasive Klebsiella pneumoniae with Hypermucoviscosity Phenotype in a Research Colony of Nonhuman Primates

Invasive Klebsiella pneumoniae with hypermucoviscosity phenotype (HMV K. pneumoniae) is an emerging human pathogen that, over the past 20 y, has resulted in a distinct clinical syndrome characterized by pyogenic liver abscesses sometimes complicated by bacteremia, meningitis, and endophthalmitis. Infections occur predominantly in Taiwan and other Asian countries, but HMV K. pneumoniae is considered an emerging infectious disease in the United States and other Western countries. In 2005, fatal multisystemic disease was attributed to HMV K. pneumoniae in African green monkeys (AGM) at our institution. After identification of a cluster of subclinically infected macaques in March and April 2008, screening of all colony nonhuman primates by oropharyngeal and rectal culture revealed 19 subclinically infected rhesus and cynomolgus macaques. PCR testing for 2 genes associated with HMV K. pneumoniae, rmpA and magA, suggested genetic variability in the samples. Random amplified polymorphic DNA analysis on a subset of clinical isolates confirmed a high degree of genetic diversity between the samples. Environmental testing did not reveal evidence of aerosol or droplet transmission of the organism in housing areas. Further research is needed to characterize HMV K. pneumoniae, particularly with regard to genetic differences among bacterial strains and their relationship to human disease and to the apparent susceptibility of AGM to this organism.Abbreviations: AGM, African green monkey; HMV K. pneumoniae, invasive Klebsiella pneumoniae with hypermucoviscosity phenotype; NHP, nonhuman primate; RAPD, random amplification of polymorphic DNAKlebsiella pneumoniae is an enteric, gram-negative, lactose-fermenting bacillus with a prominent capsule. This bacterium has been associated with peritonitis, septicemia, pneumonia, and meningitis in both Old and New World primates,^10,13,29 although it also is reported to constitute normal fecal and oral flora in many nonhuman primates (NHP).¹² Pathogenic strains associated with the upper respiratory tract typically are heavily encapsulated.¹² Over the past several decades, human medical literature indicates the emergence of an invasive K. pneumoniae disease in Taiwan and other Asian countries, in which community-acquired pyogenic liver abscesses have been attributed to strains of invasive K. pneumoniae with a unique hypermucoviscous phenotype (HMV K. pneumoniae).^{6,17-19,21,26,34} The hypermucoviscous phenotype has also been associated with other serious complications, including bacteremia, meningitis, and endophthalmitis. This strain of Klebsiella has become an emerging cause of pyogenic liver abscesses in some nonAsian countries, including the United States.^16,20,36,39 The majority of clinical cases of HMV K. pneumoniae are in the Asian population, particularly in patients with diabetes mellitus.^3,4,33 Determination of the HMV phenotype typically is based on a positive string test.^8,35,39Several virulence factors have been associated with HMV K. pneumoniae. Klebsiella spp. generally develop prominent polysaccharide capsules which increase virulence by protecting the bacteria from phagocytosis and preventing destruction by bactericidal serum factors. Capsular serotypes K1 or K2 have been reported as the major virulence determinants for human HMV K. pneumoniae liver abscesses.^5,8,37,38 In addition, the mucoviscosity-associated gene magA, which encodes a structural outer membrane protein of the K1 serotype, and rmpA (regulator of the mucoid phenotype gene; located on a plasmid) have been proposed as virulence factors.^{9,27,31,40,41} Recently, it was suggested that 2 clones, CC23 ^K1 and CC82^K1, are strongly associated with primary liver abscess and respiratory infection, respectively.²Over a period of several months in 2005 to 2006, 7 African green monkeys (AGM; Chlorocebus aethiops) in the US Army Medical Research Institute of Infectious Diseases research colony developed abscesses in multiple locations and either died or were euthanized when the abscesses were determined to be nonresectable.³⁵ HMV K. pneumoniae of the K2 serotype and carrying rmpA was determined to be the cause of the infection in 1 case, and the 6 other cases had similar clinical and pathologic features. This report³⁵ is the only documentation, to our knowledge, of natural infection with HMV K. pneumoniae in NHP. As a result of these cases, the US Army Medical Research Institute of Infectious Diseases instituted policies to exclude HMV K. pneumoniae from the colony. The organism was included as a specific pathogen-free requirement for vendors, and K. pneumoniae culture results were reported during quarantine periods and on routine semiannual examination for all colony NHP.     

Gag p27-Specific B- and T-Cell Responses in Simian Immunodeficiency Virus SIVagm-Infected African Green Monkeys

Nonpathogenic simian immunodeficiency virus SIVagm infection of African green monkeys (AGMs) is characterized by the absence of a robust antibody response against Gag p27. To determine if this is accompanied by a selective loss of T-cell responses to Gag p27, we studied CD4⁺ and CD8⁺ T-cell responses against Gag p27 and other SIVagm antigens in the peripheral blood and lymph nodes of acutely and chronically infected AGMs. Our data show that AGMs can mount a T-cell response against Gag p27, indicating that the absence of anti-p27 antibodies is not due to the absence of Gag p27-specific T cells.Simian immunodeficiency virus (SIV) infection in African green monkeys (AGM) is nonpathogenic, even though it is characterized by plasma viral load (PVL) levels similar to those found during acute and chronic pathogenic infection of humans with human immunodeficiency virus type 1 and macaques with SIVmac (14). This feature is shared with other African nonhuman primates, such as sooty mangabeys (SM) and mandrills (19, 20). SIV-infected AGMs also display high viral loads in the gastrointestinal mucosa (11), a transient decline of circulating CD4⁺ T cells during acute infection (13), and longer-lasting CD4⁺ T-cell depletion in the intestinal lamina propia (10). Concomitant with the peak viral load during acute infection, SIVagm-infected AGMs display transient increases of CD4⁺ and CD8⁺ T cells expressing activation, and proliferation markers, such as MHC-II DR and Ki-67 (4, 13), and anti-SIVagm antibodies (Ab) are induced with kinetics similar to those found in SIVmac infection (5). Interestingly, however, the Ab response against Gag p27 is weak, if present at all (1, 2, 12, 15, 17, 18). This observation is surprising since, in the context of human immunodeficiency virus type 1 and SIVmac infections, Ab responses to Gag p27 are usually quite strong. Weak or low reactivity to Gag p27 has also been observed in some other natural SIV infections (7, 8, 20) but not in all of them (21). We wondered whether such a selective lack of Ab reactivity in the SIV-infected AGM might be related to a lack of Gag p27-specific T cells. With this hypothesis in mind, we first confirmed and extended the studies of humoral responses against Gag p27 by characterizing the antigen-specific immunoglobulin G (IgG) responses and mid-point titers against total SIVagm antigens (SIVagm virions) and recombinant Gag p27 (rP27; SIVagm) in naturally and experimentally SIVagm-infected AGMs. Second, we searched for the presence of Gag p27-specific T-cell responses in SIVagm infection by analyzing the CD4⁺ and CD8⁺ T-cell responses specific for Gag p27 and other SIVagm proteins in blood and lymph nodes (LNs) of acutely and chronically infected animals.Humoral responses against SIV were analyzed in 50 wild-born AGMs (Chlorocebus sabaeus) and 17 rhesus macaques (RMs). The animals were housed at the Institut Pasteur in Dakar, Senegal, and the California National Primate Research Center, Davis, CA, respectively, according to institutional and national guidelines. RMs were either noninfected (n = 5) or intravenously infected with SIVmac251 (n = 12). AGMs were noninfected (n = 23), naturally infected (n = 17), or intravenously infected with wild-type SIVagm.sab92018 (n = 10) (5, 9). IgG titers against SIVagm.sab92018 virions or rP27 were determined by an enzyme-linked immunosorbent assay (ELISA) using monkey anti-IgG as secondary Ab (Fig. 1A and B). The virions had been purified by ultracentrifugation on an iodixanol cushion from cell-free supernatants of SIVagm.sab92018-infected SupT1 cells. The His-tagged rP27 was constructed using DNA from gut cells of an SIVagm.sab92018-infected AGM 96011 (11). A Gag p27 PCR product was subcloned into pET-14b, and the recombinant protein was produced in Escherichia coli BL21(DE3)(pLysS) and purified on nitrilotriacetic acid columns. SIV-infected macaques showed high IgG titers cross-reacting with both SIVagm virions (Fig. 1A and B, left panels) and rP27 (Fig. 1A and B, right panels). In contrast, only 2 out of 27 SIV-infected AGMs showed detectable IgG responses against rP27 (Fig. 1A and B, right panels), while 21 out of 27 displayed significant responses against SIVagm virions (Fig. 1A and B, left panels). Two AGMs out of 23 from the negative control group showed weak responses at the limit of detection against SIVagm and two against rP27, suggesting a natural response against SIVagm proteins, cross-reactivity with unknown pathogens, maternal Ab, or recent SIV infection. Of note, the titers against whole SIV in the infected monkeys were higher in macaques than in AGMs, which may be due to a lack of anti-p27 Ab in most AGMs.Open in a separate windowFIG. 1.Cross-sectional analysis of IgG Ab responses against SIVagm or Gag p27 in SIV-infected AGMs and RMs. (A and B) Cross-sectional analysis by ELISA. IgG Ab against SIVagm.sab₉₂₀₁₈ virions or recombinant p27-Gag antigens were determined in SIV-negative (Rh SIV−) and chronically SIVmac₂₅₁-infected (Rh SIV+) RMs and in SIV-negative and chronically SIVagm-infected AGMs that were either naturally (AGM Nat SIV+) or experimentally (AGM Exp SIV+) infected with SIVagm.sab₉₂₀₁₈. Ab titers were calculated for each animal by limited dilution of plasma on coated ELISA plates with 5 μg/ml of (p27 equivalent) virions (left) or 1 μg/ml of the monomeric recombinant protein (rP27) (right). IgG detection by ELISA displayed a high background for rP27, especially at the highest plasma concentration (e.g., 1/100 and 1/400 plasma dilution) in SIV-negative RMs and AGMs. To discriminate between positive responses and background, calculated dose-response curves were compared to theoretical sigmoid-dose response curves corresponding to the 95% confidence interval of SIV-negative animals. By convention, responses were considered background when sigmoid dose-response curves were graphically within the 95% confidence interval of SIV-negative animals and when the calculated negative log 50% effective concentration (EC₅₀) was lower than the top theoretical sigmoid dose-response curve from SIV-negative animals (corresponding to a threshold of negative log EC₅₀ of 2.8). (A) Results (optical density at 450 nm [OD₄₅₀]) are represented for both virions (left) and rP27 (right) over plasma dilution (log₁₀) on a per animal basis (data points) and for each group (lines). Lines represent the sigmoid dose-response curves for each group (Prism 4; Graphpad). (B) Mid-point IgG titers were determined for each animal from individual sigmoid dose-response curves, and presented as the log₁₀ value from the reciprocal of the effective concentration that corresponds to 50% response between minimum and maximum OD₄₅₀ (negative log EC₅₀). Horizontal bars represent the median mid-point titer per each group. Mann-Whitney nonparametric tests were applied for statistical analysis (n.s., nonsignificant, with P values of >0.1) (C) Cross-sectional analysis of Ab against SIVagm proteins by Western blot analysis using denatured SIVagm.sab₉₂₀₁₈. For the positive controls on the left, we used sera from an SIVmac₂₅₁-infected macaque and a SIVagm.sab₉₂₀₁₈-infected AGM. Development times and reagents were identical for all Western blots. Mo, months of infection; y, years of infection; C−, negative control; C+, positive control.The study of IgGs by Western blot analysis using denatured SIVagm.sab92018 virions showed no or weak anti-Gag responses in SIV-infected AGMs, yet the anti-Env responses were often strong (Fig. ​(Fig.1C).1C). In contrast, SIV-infected macaques showed a dominant IgG cross-reactive response against the SIVagm Gag p27 protein. Even if responses in AGMs were detected more frequently with the Western blot analyses than with the ELISAs, these responses were different in magnitude and considerably weaker than those in macaques.To compare B- and T-cell responses over time, five simian T-cell leukemia virus-seronegative AGMs were infected with SIVagm.sab92018, and the animals were followed longitudinally during the acute and postacute phases of infection until day 90 postinfection (p.i.). Sequential blood samples were collected and biopsies of auxiliary and inguinal LNs were performed on day −5 and at three times p.i. (days 14, 43, and 62). PVL was measured by real-time PCR (5). Since we searched for Gag p27-specific responses, we also quantified Gag p27 antigen in the plasma (SIV p27 antigen assay; Coulter, Miami, FL). Viral RNA and p27 antigenemia peaks were observed between days 7 and 14 p.i. (Fig. 2A and B, respectively). The Gag p27 levels were variable among the animals but in a range similar to those reported previously in AGMs and macaques (3, 5). As has also been observed in SIVmac infection (except for rapid progressors), plasma Gag p27 levels fell below the detection level in the postacute phase (i.e., after day 28 p.i.) (Fig. ​(Fig.2B2B and data not shown). There were significant increases in circulating CD8⁺ DR⁺ T cells at days 7 and 14 p.i. and in CD8⁺ Ki-67⁺ T cells at days 14 and 28 p.i. (Fig. 2C and D, left panels). After day 28 p.i., the percentages were no longer statistically different from baseline levels. In LN cells (LNCs), the percentage of CD8⁺ Ki-67⁺ T cells rose from 3.1% ± 1.1% before infection to 6.1% ± 0.3% at day 62 p.i., but the difference was not statistically significant (Fig. ​(Fig.2D,2D, right panel). The levels of blood CD4⁺ DR⁺ Ki-67⁺, CD8⁺ DR⁺ Ki-67⁺, CD8⁺ Ki-67⁺ T cells, and LNC CD8⁺ Ki-67⁺ T cells were positively correlated with viremia (P values of 0.002 for DR⁺ cells and P values of <0.02 for Ki-67⁺ cells). Altogether, these results confirm previous data showing early, transient T-cell activation in the peripheral blood of SIVagm-infected AGMs (13).Open in a separate windowFIG. 2.Plasma viremia and T-cell activation in blood and LNs of five longitudinally followed SIVagm.sab₉₂₀₁₈-infected African green monkeys. (A) SIVagm.sab RNA copy numbers in plasma. (B) Plasma Gag p27 concentrations. (C) Percentages of MHC-II DR-positive CD4⁺ (•) and CD8⁺ (○) T cells within, respectively, total CD4⁺ and CD8⁺ T cells from PBMCs and LNCs. (D) Percentages of Ki-67⁺ CD4⁺ (•) and CD8⁺ (○) T cells within, respectively, total CD4⁺ and CD8⁺ T cells from PBMCs and LNCs. Results are shown as the mean ± the standard error of the mean. Asterisks indicate statistically significant differences compared to levels before infection (P < 0.05).We next looked for the presence of Ab responses against rP27 in these animals. No Ab were detected before infection. After infection, all five AGMs developed anti-SIVagm IgGs within 4 to 9 weeks p.i., with AGM 02001 showing the fastest response (Fig. ​(Fig.3A).3A). While the humoral responses against whole virions were significant (Fig. ​(Fig.3B),3B), the anti-rP27 responses were below the threshold for positivity (Fig. ​(Fig.3B),3B), with the exception of one animal (AGM 02001). The anti-rP27 response in this animal was only transient since it was no longer detectable at week 75 p.i., in contrast to the anti-SIV Ab that were sustained (Fig. ​(Fig.3B3B and data not shown).Open in a separate windowFIG. 3.Longitudinal analysis of IgG titers and T-cell proliferative responses against SIVagm and Gag p27 in five AGMs experimentally infected with SIVagm.sab₉₂₀₁₈. (A and B) Ab responses were analyzed by ELISA. (A) IgG dose-response curves against SIVagm (top) and rP27 (bottom) are shown over time (week −1 to week 24 p.i.). O.D.450, optical density at 450 nm. (B) Mid-point titers were calculated as described in the legend to Fig. ​Fig.1A.1A. Continuous lines correspond to median titers from all five animals. Red, anti-SIVagm IgGs; green, anti-p27 IgGs. (C) Proliferative responses of CD4⁺ and CD8⁺ T cells were assessed by flow cytometry using carboxy fluorescein succinimidyl ester staining (CFSE). CD4⁺ and CD8⁺ T-cell responses in PBMCs (left) and LNCs (right) after stimulation with peptide pools (Gag without P27, P27, and Tat) and Gag rP27 are shown for each animal. All data are reported after background subtraction. Results are presented in columns as the mean ± the standard error of the mean. Asterisks indicate statistically significant differences compared to individual values before infection (P < 0.05).We next searched for T-cell responses against Gag p27 compared to other SIVagm antigens in these animals. Gag p27 epitopes were presented in the following two ways: in the context of rP27 and as synthetic peptides. The peptide pools (comprised of overlapping 15-mers) spanned the following SIVagm proteins: Gag p27, Gag without p27, Env, and Tat. The amino acid sequences of the Gag and Env peptides corresponded to the autologous wild-type SIVagm.sab92018 sequence, and those of the Tat peptides corresponded to an SIVagm.sab consensus sequence. The latter was determined using Tat sequences of other SIVagm viruses from Senegal that are available in the databases (SIVagm.sab1c, SIVagm.sabD42, and SIVagm.sabD30). We measured T-cell responses by investigating the antigen-induced proliferation. T cells from blood (peripheral blood mononuclear cells [PBMCs]) and LNs were analyzed. All assays were performed with fresh cells that were stimulated with 10 μg/ml of Gag rP27 and 5 μg/ml of peptides over a period of 4 days. Dead cells were gated out using 7-amino-actinomycin D, and dividing (CFSE^low) cells were analyzed after stimulation with medium alone, SIV antigens, or concanavalin A as a positive control. We detected significant Gag p27-specific proliferative responses for CD8⁺ T cells in PBMCs and for CD4⁺ and CD8⁺ T cells in LNCs (Fig. ​(Fig.3C).3C). The animal with the detectable anti-p27 Ab (AGM 02001) did not show stronger p27-specific T-cell responses than the other animals. Thus, all SIV-infected AGMs were able to mount a proliferative T-cell response against p27, while anti-p27 IgGs were lacking in four of the animals. However, the SIVagm-specific T-cell responses were detected at only a few time points p.i.We then analyzed the T-cell responses in the chronic phase of AGMs naturally and experimentally infected with SIVagm.sab92018. PVL, peripheral blood cell counts (CD4⁺ and CD8⁺ T cells; CD20⁺ B cells), and immune activation (Ki-67⁺ CD4⁺ and CD8⁺ T cells) were similar in naturally infected and in experimentally infected AGMs (Fig. ​(Fig.4A).4A). As expected, cell counts and immune activation levels were also not different from SIV-negative AGMs (Fig. ​(Fig.4A).4A). Again, we measured SIV-specific responses first by a proliferation assay (Fig. ​(Fig.4B).4B). One out of five animals tested had a proliferative SIV-specific CD4⁺ T-cell response (against Gag without p27, P27, rP27, Env GP120, and Tat), and two animals had a CD8⁺ T-cell response (against P27 in both animals and against Env GP120 and Tat in one). Two animals (one naturally infected and one experimentally infected with SIVagm.sab92018) did not show any detectable antigen-specific proliferative CD4⁺ or CD8⁺ T-cell response.Open in a separate windowFIG. 4.Immune parameters and SIVagm-specific proliferative and cytokine T-cell responses in chronically infected AGMs. (A) Cell counts (CD4⁺ and CD8⁺ T cells; B cells) and immune activation levels (percent of Ki-67⁺ in CD4⁺ and CD8⁺ T cells) in AGMs (n = 4) naturally infected with SIVagm (Nat SIV⁺) and AGMs (n = 6) experimentally infected with SIVagm.sab₉₂₀₁₈ (Exp SIV⁺) compared to uninfected AGMs (n = 10) (SIV⁻). PVL, if known, is indicated. Green, blue, and orange symbols correspond, respectively, to noninfected, naturally infected, and experimentally infected AGMs. (B) Proliferative response to SIVagm antigens in chronically infected AGMs (n = 5) compared to those in uninfected AGMs (n = 3). PBMCs were stimulated with the same antigens as those described in the legend to Fig. ​Fig.3.3. (C) Analysis of cytokine responses (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]) by SIVagm-specific T cells. ConA was used as a positive control. Representative results from a single animal are shown here. (D) Cumulative values of SIVagm-specific TNF-α and IFN-γ responses in chronically infected animals. The responses to SIVagm antigens were analyzed in peripheral blood specimens of 4 naturally and 5 experimentally infected AGMs as well as 10 uninfected AGMs. The data are reported after background subtraction corresponding to the subtraction of the frequency of positive events from the unstimulated samples to the frequency of positive events from the antigen-specific stimulation. Proliferative T-cell responses and cytokine T-cell responses in SIV-infected AGMs were defined as positive when higher than 3 standard deviations above the mean responses for uninfected animals. Freq, frequency; w/o, without.These results were extended to an analysis of SIV-specific T-cell cytokine responses, e.g., the production of IFN-γ and TNF-α in nine chronically infected compared to 10 noninfected AGMs (Fig. 4C and D). Fresh cells were stimulated for 8 h with the antigens described above. SIV-specific cytokine responses were detected in CD8⁺ but not in CD4⁺ T cells. Seven animals out of nine showed a response against at least one antigen. The two animals showing no response were among the four naturally infected animals tested. We therefore cannot exclude that the absence of response in these two animals is due to the presence of highly divergent viruses. However, a precise epitope mapping in SIVagm sequences would be necessary to confirm this. In those animals showing a SIVagm-specific cytokine T-cell response, the responses were directed against Gag p27 (four out of nine animals), other Gag proteins than p27 (two out of nine animals), and Env GP120 (four out of nine animals). In the experimentally infected animals, we might have underestimated the responses against Tat compared to Gag and Env antigens, since the Tat peptides corresponded to an SIVagm.sab consensus sequence and not to the autologous virus (SIVagm.sab92018). There was no correlation between the magnitude or breadth of SIV-specific T-cell responses and immune activation or PVL.Altogether, our study demonstrates that AGMs can mount T-cell proliferative and cytokine responses against Gag p27. The T-cell response was variable among the animals. In general, it appeared moderate, comparable to chronically SIV-infected RMs (9). Of note, T-cell responses were not consistently detected at all time points and not in all animals. We cannot exclude the possibility that we underestimated the magnitude of the cytokine responses. For instance, we did not costimulate the cells during the assays. However, cytokine responses were also variable in vervet AGMs, with a trend for reduced levels compared to those for RMs, even when more-sensitive assays were used (23). In SM, the responses were also reported to be not stronger than in RMs. This is in line with the lack of efficient control of viral replication in natural hosts (6, 22).In our study, we show that IgG responses against Gag p27 are either lacking, weak, or transient, while Ab against other SIVagm proteins are present. The mechanisms underlying this selective lack of Gag p27 Ab responses are unclear. It could be related to moderate and/or dysfunctional CD4⁺ T-cell responses and/or due to an unknown suppressive regulatory mechanism. SIV-specific T-cell cytokine responses were indeed principally found at the CD8⁺ T-cell level. This was also reported in SIVsm-infected SM (6, 22). Here, we also searched for SIVagm Gag p27-specific proliferative responses. Interestingly, they were detected for CD4⁺ T cells, indicating the presence of p27-specific CD4⁺ memory cells in AGMs. Moreover, AGMs can potentially mount a strong and sustained anti-Gag p27 humoral response, when appropriately immunized (D. Favre et al., unpublished data). This suggests that there is neither a central B-cell tolerance against p27 Gag protein in AGMs nor an inherent inability for CD4⁺ T cells to provide helper B-cell functions. The transient nature of anti-p27 Ab in one animal would be in favor of regulatory mechanisms, but that needs to be confirmed. Another explanation could be that AGMs are able to mount Ab responses against some p27 epitopes but not to those exposed by the native protein, which would explain why we and others detect more frequently humoral responses in Western blot analysis than in ELISAs (16).In conclusion, we characterized the IgG responses against SIVagm and confirmed a lower humoral response against p27 than in RMs. Moreover, our study reveals that cytokine and proliferative T-cell responses against SIVagm Gag p27 are detectable in AGMs. Thus, the reduced ability of the AGM to produce Ab against Gag p27 p.i. is not related to a lack of Gag p27-specific T cells.     

Enhancing the Activity of a Protein by Stereospecific Unfolding: CONFORMATIONAL LIFE CYCLE OF INSULIN AND ITS EVOLUTIONARY ORIGINS*S??

A central tenet of molecular biology holds that the function of a protein is mediated by its structure. An inactive ground-state conformation may nonetheless be enjoined by the interplay of competing biological constraints. A model is provided by insulin, well characterized at atomic resolution by x-ray crystallography. Here, we demonstrate that the activity of the hormone is enhanced by stereospecific unfolding of a conserved structural element. A bifunctional β-strand mediates both self-assembly (within β-cell storage vesicles) and receptor binding (in the bloodstream). This strand is anchored by an invariant side chain (Phe^B24); its substitution by Ala leads to an unstable but native-like analog of low activity. Substitution by d-Ala is equally destabilizing, and yet the protein diastereomer exhibits enhanced activity with segmental unfolding of the β-strand. Corresponding photoactivable derivatives (containing l- or d-para-azido-Phe) cross-link to the insulin receptor with higher d-specific efficiency. Aberrant exposure of hydrophobic surfaces in the analogs is associated with accelerated fibrillation, a form of aggregation-coupled misfolding associated with cellular toxicity. Conservation of Phe^B24, enforced by its dual role in native self-assembly and induced fit, thus highlights the implicit role of misfolding as an evolutionary constraint. Whereas classical crystal structures of insulin depict its storage form, signaling requires engagement of a detachable arm at an extended receptor interface. Because this active conformation resembles an amyloidogenic intermediate, we envisage that induced fit and self-assembly represent complementary molecular adaptations to potential proteotoxicity. The cryptic threat of misfolding poses a universal constraint in the evolution of polypeptide sequences.How insulin binds to the insulin receptor (IR)² is not well understood despite decades of investigation. The hormone is a globular protein containing two chains, A (21 residues) and B (30 residues) (Fig. 1A). In pancreatic β-cells, insulin is stored as Zn²⁺-stabilized hexamers (Fig. 1B), which form microcrystal-line arrays within specialized secretory granules (1). The hexamers dissociate upon secretion into the portal circulation, enabling the hormone to function as a zinc-free monomer. The monomer is proposed to undergo a change in conformation upon receptor binding (2). In this study, we investigated a site of conformational change in the B-chain (Phe^B24) (arrow in Fig. 1A). In classical crystal structures, this invariant aromatic side chain (tawny in Fig. 1B) anchors an antiparallel β-sheet at the dimer interface (blue in Fig. 1C). Total chemical synthesis is exploited to enable comparison of corresponding d- and l-amino acid substitutions at this site, an approach designated “chiral mutagenesis” (3-5). In the accompanying article, the consequences of this conformational change are investigated by photomapping of the receptor-binding surface (6). Together, these studies redefine the interrelation of structure and activity in a protein central to the hormonal control of metabolism.Open in a separate windowFIGURE 1.Sequence and structure of insulin. A, sequences of the B-chain (upper) and A-chain (lower) with disulfide bridges as indicated. The arrow indicates invariant Phe^B24. The B24-B28 β-strand is highlighted in blue. B, crystal structure of the T₆ zinc insulin hexamer (Protein Data Bank code 4INS): ribbon model (left) and space-filling model (right). The B24-B28 β-strand is shown in blue, and the side chain of Phe^B24 is highlighted in tawny. The B-chain is otherwise dark gray; the A-chain, light gray; and zinc ions, magenta. Also shown at the left are the side chains of His^B10 at the axial zinc-binding sites. C, cylinder model of the insulin dimer showing the B24-B26 antiparallel β-sheet (blue) anchored by the B24 side chain (tawny circle). The A- and B-chains are shown in light and dark gray, respectively. The protomer at the left is shown in the R-state, in which the central α-helix of the B-chain is elongated (B3-B19 in the frayed R^f protomer of T₃R^f₃ hexamers and B1-B19 in the R protomer of R₆ hexamers). The three types of zinc insulin hexamers share similar B24-B26 antiparallel β-sheets as conserved dimerization elements.The structure of an insulin monomer in solution resembles a crystallographic protomer (Fig. 2A) (7-9). The A-chain contains an N-terminal α-helix, non-canonical turn, and second helix; the B-chain contains an N-terminal segment, central α-helix, and C-terminal β-strand. The β-strand is maintained in an isolated monomer wherein the side chain of Phe^B24 (tawny in Fig. 2A), packing against the central α-helix of the B-chain, provides a “plug” to seal a crevice in the hydrophobic core (Fig. 2B). Anomalies encountered in previous studies of insulin analogs suggest that Phe^B24 functions as a conformational switch (4, 7, 10-14). Whereas l-amino acid substitutions at B24 generally impair activity (even by such similar residues as l-Tyr) (15), a seeming paradox is posed by the enhanced activities of nonstandard analogs containing d-amino acids (10-12).

TABLE 1

Previous studies of insulin analogs

Flowering time regulation by the SUMO E3 ligase SIZ1

Flowering is a developmental process, which is influenced by chemical and environmental stimuli. Recently, our research established that the Arabidopsis SUMO E3 ligase, AtSIZ1, is a negative regulator of transition to flowering through mechanisms that reduce salicylic acid (SA) accumulation and involve SUMO modification of FLOWERING LOCUS D (FLD). FLD is an autonomous pathway determinant that represses the expression of FLOWERING LOCUS C (FLC), a floral repressor. This addendum postulates mechanisms by which SIZ1-mediated SUMO conjugation regulates SA accumulation and FLD activity.Key words: SIZ1, SA, flowering, SUMO, FLD, FLCSUMO conjugation and deconjugation are post-translational processes implicated in plant defense against pathogens, abscisic acid (ABA) and phosphate (Pi) starvation signaling, development, and drought and temperature stress tolerance, albeit only a few of the modified proteins have been identified.^1–8 The Arabidopsis AtSIZ1 locus encodes a SUMO E3 ligase that regulates floral transition and leaf development.^8,9 siz1 plants accumulate substantial levels of SA, which is the primary cause for dwarfism and early short-day flowering exhibited by these plants.^1,9 How SA promotes transition to flowering is not yet known but apparently, it is through a mechanism that is independent of the known floral signaling pathways.^9,10 Exogenous SA reduces expression of AGAMOUS-like 15 (AGL15), a floral repressor that functions redundantly with AGL18.^11,12 A possible mechanism by which SA promotes transition to flowering may be by repressing expression of AGL15 and AGL18 (Fig. 1).Open in a separate windowFigure 1Model of how SUMO conjugation and deconjugation regulate plant development in Arabidopsis. SIZ1 and Avr proteins regulate biosynthesis and accumulation of SA, a plant stress hormone that is involved in plant innate immunity, leaf development and regulation of flowering time. SA promotes transition to flowering may through AGL15/AGL18 dependent and independent pathways. FLC expression is activated by FRIGIDA but repressed by the autonomous pathway gene FLD, and SIZ1-mediated sumoylation of FLD represses its activity. Lines with arrows indicate upregulation (activation), and those with bars identify downregulation (repression).siz1 mutations also cause constitutive induction of pathogenesis-related protein genes leading to enhanced resistance against biotrophic pathogens.¹ Several bacterial type III effector proteins, such as YopJ, XopD and AvrXv4, have SUMO isopeptidase activity.^13–15 PopP2, a member of YopJ/AvrRxv bacterial type III effector protein family, physically interacts with the TIR-NBS-LRR type R protein RRS1, and possibly stabilizes the RRS1 protein.¹⁶ Phytopathogen effector and plant R protein interactions lead to increased SA biosynthesis and accumulation, which in turn activates expression of pathogenesis-related proteins that facilitate plant defense.¹⁷ SIZ1 may participate in SUMO conjugation of plant R proteins to regulate Avr and R protein interactions leading to SA accumulation, which, in turn, affects phenotypes such as diseases resistance, dwarfism and flowering time (Fig. 1).Our recent work revealed also that AtSIZ1 facilitates FLC expression, negatively regulating flowering.⁹ AtSIZ1 promotes FLC expression by repressing FLD activity.⁹ Site-specific mutations that prevent SUMO1/2 conjugation to FLD result in enhanced activity of the protein to represses FLC expression, which is associated with reduced acetylation of histone 4 (H4) in FLC chromatin.⁹ FLD, an Arabidopsis ortholog of Lysine-Specific Demethylase 1 (LSD1), is a floral activator that downregulates methylation of H3K4 in FLC chromatin and represses FLC expression.^18,19 Interestingly, bacteria expressing recombinant FLD protein did not demethylate H3K4me2, inferring that the demethylase activity requires additional co-factors as are necessary for LSD1.^18,20 Together, these results suggest that SIZ1-mediated SUMO modification of FLD may affect interactions between FLD and co-factors, which is necessary for FLC chromatin modification.Despite our results that implicate SA in flowering time control, how SIZ1 regulates SA accumulation and the identity of the effectors involved remain to be discovered. In addition, it remains to be determined if SIZ1 is involved in other mechanisms that modulate FLD activity and FLC expression, or the function of other autonomous pathway determinants.     

Induction of systemic resistance in rice by leaf extracts of Zizyphus jujuba and Ipomoea carnea against Rhizoctonia solani

Plants accumulate a great diversity of natural products, many of which confer protective effects against phytopathogenic attack. Earlier we had demonstrated that the leaf extracts of Zizyphus jujuba and Ipomoea carnea inhibit the in vitro mycelial growth of Rhizoctonia solani, and effectively reduce the incidence of sheath blight disease in rice.⁷ Here we demonstrate that foliar application of the aqueous leaf extracts of Z. jujuba and I. carnea followed by challenge inoculation with R. solani induces systemic resistance in rice as evident from significantly increased accumulation of pathogenesis-related proteins such as chitinase, β-1,3-glucanase and peroxidase, as well as defense-related compounds such as phenylalanine ammonia-lyase and phenolic substances. Thin layer chromatographic separation of secondary metabolites revealed presence of alkaloid and terpenoid compounds in the leaf extracts of Z. jujuba that exhibited toxicity against R. solani under in vitro condition. Thus, the enhanced sheath blight resistance in rice seedlings treated with leaf extracts of Z. jujuba or I. carnea can be attributed to the direct inhibitory effects of these leaf extracts as well as their ability to elicit systemic resistance against R. solani.Key words: sheath blight, Zizyphus jujuba, Ipomoea carnea, Rhizoctonia solani, induced systemic resistance, antimicrobial compoundsSheath blight disease of rice, caused by Rhizoctonia solani, has become a major production constraint in intensive rice cropping systems where semi-dwarf, nitrogen-responsive and high-yielding rice cultivars are grown. The disease causes an annual yield loss of upto 50%.¹ R. solani is both soil- and water-borne, and can infect more than 27 families of both monocot and dicot species.² Natural host genetic resistance to R. solani has not been recorded in cultivars or wild relatives of rice.³ Several broad spectrum fungicides have been recommended for control of sheath blight, however, chemical method of disease management is neither practical due to high cost of fungicides nor sustainable as it can affect the balance of ecosystem by destroying beneficial microbial population. In addition, the environmental pollution problems associated with indiscriminate use of synthetic pesticides have prompted investigations on exploiting bio-pesticides of plant and microbial origin.Plants accumulate an enormous variety of over 100,000 secondary metabolites,⁴ which can act as pre-existing chemical inhibitors to invading pathogens and/or help strengthen defense response of host plant. The pre-formed infectional barriers in plants are generally referred to as “phytoanticipins;” whereas, the antimicrobial compounds that are synthesized de novo in response to pathogen attack are referred to as “phytoalexins.”⁵ Because of years of selective breeding leading to removal of natural products, the endogenous levels of phytoanticipins in commonly cultivated crop species are generally low and often not sufficient to fight pathogen attack, effectively.⁴ Various weed species and wild relatives of crop plants that are not subjected to selective breeding are believed to contain higher levels of antimicrobial compounds, consistent with their ability to fight invading pathogens more effectively than cultivated crop species. Identification of such weed/plant species that are enriched with antimicrobial principles, isolation of bio-active compounds from them, and application in the form of concentrated formulations to crop plants can augment their disease resistance capability by directly inhibiting the growth of pathogen and inducing defense responses. Indeed, the antimicrobial properties of tissue extracts of several weed/plant species have been reported by a number of research groups world-wide, especially in Asia and Latin America.^6–13Earlier, we had evaluated the antimicrobial activity of leaf extracts of 16 different plant species belonging to 16 different families and demonstrated that leaf extracts of most of these plant species exhibit growth-inhibitory activities against R. solani and Xanathomoas oryzae pv. oryzae (Xoo).⁷ Among these, the leaf extracts of Datura metel were found to be the most effective in inhibiting the mycelial growth and sclerotia formation of R. solani, and the growth of Xoo, as well as in reducing the incidence of sheath blight and bacterial blight diseases caused by these pathogens, respectively, under greenhouse condition.⁷ We further demonstrated that rice seedlings treated with leaf extracts of D. metel accumulated significantly higher levels of pathogenesis-related (PR) proteins and other defense related compounds following challenge inoculation with R. solani or Xoo.⁷ Our attempts to identify biologically active compounds from D. metel revealed the presence of a withanolide compound “daturilin” that exhibited remarkable antibacterial activity against Xoo.⁷Apart from D. metel, two other plants species, Zizyphus jujuba and Ipomoea carnea, were found to possess remarkable antifungal activity against R. solani.⁷ Z. jujuba is a thorny rhamnaceous plant that is widely distributed in Europe and South-eastern Asia. I. carnea of convolvulaceae family, commonly known as morning glory, is a toxic weed found in abundance in India, Brazil, the United States and other countries.¹⁴ Both of these plant species have allelopathic effect and are commonly used in folklore medicine for curing multiple diseases.^15–18 The aqueous and methanol leaf extracts of Z. jujuba and I. carnea have been found to be highly effective in reducing in vitro mycelial growth, and therefore, sclerotia production of R. solani.⁷ In the greenhouse experiments, rice seedlings sprayed with leaf extracts of Z. jujuba and I. carnea exhibited 44 and 34% reduction in severity of sheath blight disease over the control, respectively.⁷ While these findings are encouraging, the mechanisms by which the leaf extracts of Z. jujuba and I. carnea modulate defense responses in rice have not yet been explored.Plants are endowed with defense genes which remain quiescent or are expressed at basal levels in healthy plants. Activation of defense genes results in induction of systemic resistance in host plant; this defense response, designated as induced systemic resistance (ISR), plays an important role in development of disease resistance.¹⁹ The onset of ISR in plants correlates with accumulation of phytoalexins and increased activity of PR proteins such as chitinases, β-1,3-glucanases and peroxidases;^20–23 consequently, PR proteins are generally used as ISR markers.¹⁹ The classical inducers of ISR include both biotic and abiotic factors, including disease causing microorganisms themselves,^24,25 plant growth promoting rhizobacteria,^22,26 chemicals^27,28 and natural plant products.^{7,10,12,13,29,30} Plant products have been considered as one of the major groups of compounds that induce ISR. To date, extracts of at least a few plant species have been reported to contain allelopathic substances which can act as elicitors and induce systemic resistance in host plants resulting in reduction or inhibition of disease development.^7,10,12,13In the present study, with the objective of understanding the mechanisms of disease suppression by leaf extracts of Z. jujuba and I. carnea, we investigated their ability to induce ISR in rice by analyzing the activities of ISR markers including PR-proteins and other defense enzymes involved in phenylpropanoid metabolism. The changes in activities of chitinase, β-1,3-glucanase, peroxidase, phenylalanine ammonia-lyase (PAL) and phenolic compounds induced in rice seedlings that were elicited with leaf extracts (at 1:10 dilution; w/v) of Z. jujuba or I. carnea and infected with R. solani were analyzed, and compared to changes in non-elicited and uninfected seedlings. Rice seedlings that were both elicited with leaf extracts of Z. jujuba or I. carnea and infected with R. solani accumulated significantly higher levels (2–5-fold) of ISR markers as compared to non-elicited and/or uninfected seedlings (Fig. 1). About two-fold increase in activities of ISR markers was also observed in seedlings that were either infected but not elicited or elicited but not infected; however, this increase was significantly lower than the changes in seedlings that were both elicited and infected (Fig. 1). Although the activity of all ISR markers began to increase around or after 24 h post-infection, at least two distinct induction patterns were observed. For instance, the activities of chitinase and phenolic substances gradually increased to reach maximum levels at 164 h post-infection (Fig. 1A and E); whereas, the activities of β-1,3-glucanase, peroxidase and PAL reached maximum levels at 72 to 96 h post-infection and decreased thereafter (Fig. 1B–D). The leaf extracts of Z. jujuba were found slightly more effective in inducing ISR markers than the leaf extracts of I. carnea. There was no significant change in the activity of ISR markers in control seedlings sprayed with sterile distilled water (Fig. 1). Collectively, these results suggested that the leaf extracts of Z. jujuba and I. carnea have the ability to induce systemic resistance in rice seedlings infected with R. solani. The fungitoxicity of the leaf extracts of Z. jujuba and I. carnea ⁷ combined with their ability to elicit ISR is possibly responsible for low sheath blight disease incidence observed in rice seedlings treated with these leaf extracts.⁷Open in a separate windowFigure 1Activity of ISR markers and defense-related compounds in rice seedlings elicited with the leaf extracts of Zizyphus jujuba or Ipomoea carnea and challenge inoculated with Rhizoctonia solani. Total activity of chitinase (A), β-1,3-glucanase (B), peroxidase (C) phenylalanine ammonia-lyase (PAL; D) and phenolic substances (E) was analyzed in rice seedlings. The inoculation of rice seedlings with R. solani was performed 45 days after planting. Spraying of leaf extracts (1:10 dilution; w/v) of Z. jujuba or I. carnea was performed two days prior to inoculation. Tissue samples (sheath) from elicited and/or infected seedlings were collected for analysis at various time intervals.The in vitro antimicrobial and in vivo disease inhibitory effects of natural plant products are generally attributed to the allelopathic substances present in them. However, very few attempts have been made to purify and characterize active principles from bio-active natural plant products. We have previously identified a withanolide compound from leaf extracts of D. metel which exhibited antibacterial activity against Xoo.⁷ Both Z. jujuba and I. carnea are rich source of secondary metabolites including alkaloids, terpenoids, flavonoids and phenolic compounds.^31–35 To determine the composition of bio-active ingredients within the leaf extracts of Z. jujuba and I. carnea, we performed thin layer chromatographic separation of alkaloid, terpenoid and phenolic compounds. The partially purified compounds, as reported in

Exocyst Complex Member EXOC5 Is Required for Survival of Hair Cells and Spiral Ganglion Neurons and Maintenance of Hearing

The exocyst, an octameric protein complex consisting of Exoc1 through Exoc8, was first determined to regulate exocytosis by targeting vesicles to the plasma membrane in yeast to mice. In addition to this fundamental role, the exocyst complex has been implicated in other cellular processes. In this study, we investigated the role of the exocyst in cochlear development and hearing by targeting EXOC5, a central exocyst component. Deleting Exoc5 in the otic epithelium with widely used Cre lines resulted in early lethality. Thus, we generated two different inner ear-specific Exoc5 knockout models by crossing Gfi1^Cre mice with Exoc5^f/f mice for hair cell-specific deletion (Gfi1^Cre/+;Exoc5^f/f) and by in utero delivery of rAAV-iCre into the otocyst of embryonic day 12.5 for deletion throughout the otic epithelium (rAAV2/1-iCre;Exoc5^f/f). Gfi1^Cre/+;Exoc5^f/f mice showed relatively normal hair cell morphology until postnatal day 20, after which hair cells underwent apoptosis accompanied by disorganization of stereociliary bundles, resulting in progressive hearing loss. rAAV2/1-iCre;Exoc5^f/f mice exhibited abnormal neurite morphology, followed by apoptotic degeneration of spiral ganglion neurons (SGNs) and hair cells, which led to profound and early-onset hearing loss. These results demonstrate that Exoc5 is essential for the normal development and survival of cochlear hair cells and SGNs, as well as the functional maintenance of hearing.     

HrpW of Erwinia amylovora,a New Harpin That Contains a Domain Homologous to Pectate Lyases of a Distinct Class

Harpins, such as HrpN of Erwinia amylovora, are extracellular glycine-rich proteins that elicit the hypersensitive reaction (HR). We identified hrpW of E. amylovora, which encodes a protein similar to known harpins in that it is acidic, rich in glycine and serine, and lacks cysteine. A putative HrpL-dependent promoter was identified upstream of hrpW, and Western blot analysis of hrpL mutants indicated that the production of HrpW is regulated by hrpL. HrpW is secreted via the Hrp (type III) pathway based on analysis of wild-type strains and hrp secretion mutants. When infiltrated into plants, HrpW induced rapid tissue collapse, which required active plant metabolism. The HR-eliciting activity was heat stable and protease sensitive. Thus, we concluded that HrpW is a new harpin. HrpW of E. amylovora consists of two domains connected by a Pro and Ser-rich sequence. A fragment containing the N-terminal domain was sufficient to elicit the HR. Although no pectate lyase activity was detected, the C-terminal region of HrpW is homologous to pectate lyases of a unique class, suggesting that HrpW may be targeted to the plant cell wall. Southern analysis indicated that hrpW is conserved among several Erwinia species, and hrpW, provided in trans, enhanced the HR-inducing ability of a hrpN mutant. However, HrpW did not increase the virulence of a hrpN mutant in host tissue, and hrpW mutants retained the wild-type ability to elicit the HR in nonhosts and to cause disease in hosts.Most gram-negative plant-pathogenic bacteria contain clusters of genes termed hrp that are required for elicitation of a rapid localized defense response called the hypersensitive reaction (HR) in incompatible plants and that are required for pathogenicity in susceptible plants (1). Proteins encoded by hrp genes are involved in the regulation of the expression of other hrp genes and in a specialized secretion process called the Hrp or type III pathway (9). Harpins, a major class of proteins that travel the pathway (including HrpN of Erwinia species, HrpZ of Pseudomonas syringae, and PopA of Ralstonia solanacearum), elicit the HR when infiltrated into the apoplast of leaf tissue (reference 1 and references therein). They are heat stable, rich in Gly and/or Ser, lack Cys, and differ in their primary sequences. In Erwinia amylovora, mutation of hrpN results in substantially reduced Hrp phenotype (4, 6, 45).E. amylovora causes the devastating fire-blight disease on many rosaceous plants, such as apple, pear, and cotoneaster. Cosmids pCPP430 and pCPP450, which harbor the hrp gene cluster of E. amylovora Ea321, enable Escherichia coli to elicit the HR in tobacco (7). The region of pCPP430 essential for the Hrp phenotype encodes two-component regulatory proteins, a ς⁵⁴ enhancer-binding protein, a sigma factor, secretory proteins, and the HrpN harpin (11, 27, 42–45). In contrast, the locus next to hrp genes, designated dsp, contains pathogenicity genes, and P. syringae pv. glycinea containing the E. amylovora dsp locus causes the HR rather than disease in soybean plants (10). This locus encodes a Hrp-secreted protein and a probable chaperone of the secreted protein (8, 10, 17).Additional HR elicitors in E. amylovora have been suspected based on the HR-variable phenotype of E. amylovora hrpN mutants (references 4 and 6; see also Table ​Table1).1). We report here the identification and characterization of a novel harpin of E. amylovora, HrpW, the C-terminal domain of which surprisingly is homologous to fungal pectate lyases (PLs). We show that HrpW, the production of which is controlled by hrpL, is delivered by the E. amylovora Hrp pathway. HrpW elicits the HR in plants, and the HR necrosis is not due to the potential PL activity of HrpW. Finally, we provide evidence that HrpW is not required for the HR and pathogenicity, although when overexpressed it enhances the HR-eliciting activity of a hrpN mutant. Preliminary reports on E. amylovora HrpW have been made (28, 29), and, while this article was under revision, a paper describing HrpW from E. amylovora CFBP1430 (16) appeared.

TABLE 1

HR induction and virulence of E. amylovora Ea321 and mutant derivatives^a

The effect of genetic environment on locus-specific instability of the <Emphasis Type="Italic">yellow</Emphasis> gene in the Uman’ population of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The effects of genotype of the laboratory strains, C(1)DX, ywf/Y, 23.5 MRF/CyL ⁴, and C(1)DX,yf; π₂, on locus-specific instability in the yellow gene of the strains y ^2-217, y ^2-715, and y ^2-700 from Uman’ population of Drosophila melanogaster was studied. Crosses of the males from Uman’-derived lines with the C(1)DX,ywf/Y females yielded a cascade of derivatives, mostly consisting of y ⁺ and y ² alleles, while their crosses with the 23.5 MRF/CyL ⁴ and C(1)DX,yf; π₂ females mostly resulted in the appearance of y ⁺ and y ¹ derivatives. The genomes of laboratory strains used in the study contained the full-sized hobo elements, which could differ from one another relative to the structure of variable region and affinity to different DNA sequences.