Crystal Structures of Cif from Bacterial Pathogens Photorhabdus luminescens and Burkholderia pseudomallei

A pre-requisite for bacterial pathogenesis is the successful interaction of a pathogen with a host. One mechanism used by a broad range of Gram negative bacterial pathogens is to deliver effector proteins directly into host cells through a dedicated type III secretion system where they modulate host cell function. The cycle inhibiting factor (Cif) family of effector proteins, identified in a growing number of pathogens that harbour functional type III secretion systems and have a wide host range, arrest the eukaryotic cell cycle. Here, the crystal structures of Cifs from the insect pathogen/nematode symbiont Photorhabdus luminescens (a γ-proteobacterium) and human pathogen Burkholderia pseudomallei (a β-proteobacterium) are presented. Both of these proteins adopt an overall fold similar to the papain sub-family of cysteine proteases, as originally identified in the structure of a truncated form of Cif from Enteropathogenic E. coli (EPEC), despite sharing only limited sequence identity. The structure of an N-terminal region, referred to here as the ‘tail-domain’ (absent in the EPEC Cif structure), suggests a surface likely to be involved in host-cell substrate recognition. The conformation of the Cys-His-Gln catalytic triad is retained, and the essential cysteine is exposed to solvent and addressable by small molecule reagents. These structures and biochemical work contribute to the rapidly expanding literature on Cifs, and direct further studies to better understand the molecular details of the activity of these proteins.     

Porin Involvement in Cephalosporin and Carbapenem Resistance of Burkholderia pseudomallei

Background

Burkholderia pseudomallei (Bps) is a Gram-negative bacterium that causes frequently lethal melioidosis, with a particularly high prevalence in the north and northeast of Thailand. Bps is highly resistant to many antimicrobial agents and this resistance may result from the low drug permeability of outer membrane proteins, known as porins.

Principal Findings

Microbiological assays showed that the clinical Bps strain was resistant to most antimicrobial agents and sensitive only to ceftazidime and meropenem. An E. coli strain defective in most porins, but expressing BpsOmp38, exhibited considerably lower antimicrobial susceptibility than the control strain. In addition, mutation of Tyr119, the most prominent pore-lining residue in BpsOmp38, markedly altered membrane permeability, substitution with Ala (mutant BpsOmp38Y119A) enhanced uptake of the antimicrobial agents, while substitution with Phe (mutant BpsOmp38Y119F) inhibited uptake. Channel recordings of BpsOmp38 reconstituted in a planar black lipid membrane (BLM) suggested that the higher permeability of BpsOmp38Y119A was caused by widening of the pore interior through removal of the bulky side chain. In contrast, the lower permeability of BpsOmp38Y119F was caused by introduction of the hydrophobic side chain (Phe), increasing the ‘greasiness’ of the pore lumen. Significantly, liposome swelling assays showed no permeation through the BpsOmp38 channel by antimicrobial agents to which Bps is resistant (cefoxitin, cefepime, and doripenem). In contrast, high permeability to ceftazidime and meropenem was observed, these being agents to which Bps is sensitive.

Conclusion/Significance

Our results, from both in vivo and in vitro studies, demonstrate that membrane permeability associated with BpsOmp38 expression correlates well with the antimicrobial susceptibility of the virulent bacterium B. pseudomallei, especially to carbapenems and cephalosporins. In addition, substitution of the residue Tyr119 affects the permeability of the BpsOmp38 channel to neutral sugars and antimicrobial agents.     

Airborne Transmission of Melioidosis to Humans from Environmental Aerosols Contaminated with B. pseudomallei

Melioidosis results from an infection with the soil-borne pathogen Burkholderia pseudomallei, and cases of melioidosis usually cluster after rains or a typhoon. In an endemic area of Taiwan, B. pseudomallei is primarily geographically distributed in cropped fields in the northwest of this area, whereas melioidosis cases are distributed in a densely populated district in the southeast. We hypothesized that contaminated cropped fields generated aerosols contaminated with B. pseudomallei, which were carried by a northwesterly wind to the densely populated southeastern district. We collected soil and aerosol samples from a 72 km² area of land, including the melioidosis-clustered area and its surroundings. Aerosols that contained B. pseudomallei-specific TTSS (type III secretion system) ORF2 DNA were well distributed in the endemic area but were rare in the surrounding areas during the rainy season. The concentration of this specific DNA in aerosols was positively correlated with the incidence of melioidosis and the appearance of a northwesterly wind. Moreover, the isolation rate in the superficial layers of the contaminated cropped field in the northwest was correlated with PCR positivity for aerosols collected from the southeast over a 2-year period. According to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analyses, PFGE Type Ia (ST58) was the predominant pattern linking the molecular association among soil, aerosol and human isolates. Thus, the airborne transmission of melioidosis moves from the contaminated soil to aerosols and/or to humans in this endemic area.     

Analysis of the Prevalence,Secretion and Function of a Cell Cycle-Inhibiting Factor in the Melioidosis Pathogen Burkholderia pseudomallei

Enteropathogenic and enterohaemorrhagic Escherichia coli express a cell cycle-inhibiting factor (Cif), that is injected into host cells via a Type III secretion system (T3SS) leading to arrest of cell division, delayed apoptosis and cytoskeletal rearrangements. A homologue of Cif has been identified in Burkholderia pseudomallei (CHBP; Cif homologue in B. pseudomallei; BPSS1385), which shares catalytic activity, but its prevalence, secretion and function are ill-defined. Among 43 available B. pseudomallei genome sequences, 33 genomes (76.7%) harbor the gene encoding CHBP. Western blot analysis using antiserum raised to a synthetic CHBP peptide detected CHBP in 46.6% (7/15) of clinical B. pseudomallei isolates from the endemic area. Secretion of CHBP into bacterial culture supernatant could not be detected under conditions where a known effector (BopE) was secreted in a manner dependent on the Bsa T3SS. In contrast, CHBP could be detected in U937 cells infected with B. pseudomallei by immunofluorescence microscopy and Western blotting in a manner dependent on bsaQ. Unlike E. coli Cif, CHBP was localized within the cytoplasm of B. pseudomallei-infected cells. A B. pseudomallei chbP insertion mutant showed a significant reduction in cytotoxicity and plaque formation compared to the wild-type strain that could be restored by plasmid-mediated trans-complementation. However, there was no defect in actin-based motility or multinucleated giant cell formation by the chbP mutant. The data suggest that the level or timing of CHBP secretion differs from a known Bsa-secreted effector and that CHBP is required for selected virulence-associated phenotypes in vitro.     

Host Range and Ecology of Isolates of Pasteuria spp. from the Southeastern United States

Isolates of Pasteuria penetrans were evaluated for ecological characteristics that are important in determining their potential as biological control agents. Isolate P-20 survived without loss of its ability to attach to its host nematode in dry, moist, and wet soil and in soil wetted and dried repeatedly for 6 weeks. Some spores moved 6.4 cm (the maximum distance tested) downward in soil within 3 days with percolating water. The isolates varied greatly in their attachment to different nematode species and genera. Of five isolates tested in spore-infested soil, three (P-104, P-122, B-3) attached to two or more nematode species, whereas B-8 attached only to Meloidogyne hapla and B-I did not attach to any of the nematodes tested. In water suspensions, spores of isolate P-20 attached readily to M. arenaria but only a few spores attached to other Meloidogyne spp. Isolate P-104 attached to all Meloidogyne spp. tested but not to Pratylenchus scribneri. Isolate B-4 attached to all species of Meloidogyne and Pratylenchus tested, but the rate of attachment was relatively low. Isolate P-Z00 attached in high numbers to M. arenaria when spores were extracted from females of this nematode; when extracted from M. javanica females, fewer spores attached to M. arenaria than to M. javanica or M. incognita.     

Vancomycin-Resistant Enterococcus spp. Isolated from Wastewater and Chicken Feces in the United States

Vancomycin-resistant Enterococcus spp. (VRE) were isolated from sewage and chicken feces but not from other animal fecal sources (dog, cow, and pig) or from surface waters tested. VRE from hospital wastewater were resistant to ≥20 μg of vancomycin/ml and possessed the vanA gene. VRE from residential wastewater and chicken feces were resistant to 3 to 5 μg of vancomycin/ml and possessed the vanC gene.     

Morphological Comparisons Between Xiphinema rivesi Daimasso and X. americanum Cobb Populations from the Eastern United States

Though in the past Xiphinema americanum has been the most commonly reported dagger nematode in the eastern United States, our studies revealed the presence in Pennsvlvania of a previously unrecognized and unreported species related to X. americanum, Morphometric data and photomicrographs establish the identity of this form as X. rivesi and show expected variations in populations of this species from various locations. Similar data and illustrations are given for X. americanum populations from Pennsylvania and other areas, showing variations and relationships. Xiphinema rivesi is widely distributed in the fruit producing area of south-central Pennsylvania and is also reported herein from raspberry in Vermont and apple in Maryland and New York. This species is frequently found in fruit growing areas of Pennsylvania associated with tomato ringspot virus-induced diseases and is also found associated with corn, bluegrass sod, and alfalfa.     

Evidence of susceptibility to morbillivirus infection in cetaceans from the United States

Cetacean morbilliviruses (CeMV) are viruses that can cause mass mortalities among various odontocete species. In this study levels of “herd” immunity in cetaceans from the U.S. coast are described from the distribution and prevalence of antibodies against morbilliviruses. Neutralizing antibody titers against dolphin morbillivirus (DMV), porpoise morbillivirus (PMV), phocine distemper (PDV), and canine distemper viruses (CDV) were measured. Positive samples had higher titers against the CeMV than against the other morbilliviruses tested, indicating that although PDV or CDV can be used to investigate exposure their use may result in a higher false negative rate. The results suggest that morbillivirus did not persist in coastal populations of bottlenose dolphins (Tursiops truncatus) after the major outbreaks that occurred in the 1980s and 1990s. Bottlenose dolphins from Beaufort, North Carolina; St. Joseph Bay, Florida; and Cape May, New Jersey had anti‐DMV seroprevalences ranging from between 15% and 33% but those from Charleston, South Carolina and Sarasota Bay, Florida, sampled in recent years were largely negative. These latter groups are therefore now vulnerable to infection and could experience high mortality if exposed to CeMV. Sero‐surveys of this kind are therefore vital for assessing the risk of new and recurring viral outbreaks in coastal cetaceans.     

Incidence and Distinguishing Characteristics of Meloidogyne chitwoodi and M. hapla in Potato from the Northwestern United States

From September 1980 to June 1981, a survey was conducted in the major potato growing regions of northern California, Idaho, Nevada, Oregon. and Washington to determine the distribution of Meloidogyne chitwoodi and other Meloidogyne spp. Meloidogyne chitwoodi and M. hapla were the only root-knot nematode species detected parasitizing potato in all the states surveyed. Meloidogyne chitwoodi occurred alone in 83% of the samples and M. hapla in 11%, with 6% of all samples containing both species. The greater incidence of M. chitwoodi, as compared to M. hapla, may be due to the cool growing season encountered in 1980 (which favored M. chitwoodi but not M. hapla) and to the increased acreage of small grains (which are good hosts for M. chitwoodi but not M. hapla) planted in rotation with potato. Differentiation between these two species can be determined by a differential host test, perineal patterns of mature females, and shape of the tail tip amt of the tail hypodermal terminus of L₂ juveniles.     

Diverse uncultivated bacterial groups from soils of the arid southwestern United States that are present in many geographic regions.

We have performed a phylogenetic survey of microbial species present in two soils from northern Arizona. Microbial DNA was purified directly from soil samples and subjected to PCR amplification with primers specific for bacterial 16S rRNA gene sequences (rDNAs). Clone libraries from the two soils were constructed, and 60 clone inserts were partially sequenced. Phylogenetic analysis of these sequences revealed extensive diversity. Most of the analyzed sequences (64%) fell into five novel clusters having no known cultured members. Extensive analysis of 10 nearly full-length rDNAs from clones representative of the novel groups indicated that four of the five groups probably cluster into a large "supergroup" which is as distinct from currently recognized bacterial divisions as the latter are from each other. From this we postulate the existence of a major bacterial lineage, previously known only from a single cultured representative, whose diversity and ecology we are only beginning to explore. Analysis of our data and that from other rDNA sequence-based studies of soils from different geographic regions shows considerable overlap of sequence types. Taken together, these groups encompass most of the novel rDNA sequences recovered in each comparable analysis reported to date, despite large differences in soil types and geographic sources. Our results indicate that members of these new groups comprise a phylogenetically diverse, geographically widespread, and perhaps numerically important component of the soil microbiota.     

Survey of Infectious Multiple Drug Resistance Among Salmonella Isolated from Animals in the United States

Salmonella cultures were obtained from outbreaks of animal disease from 37 states and 1 territory. They were screened for resistance to 11 antimicrobial drugs. Of the 1,251 strains studied, 935 were resistant to one or more of these agents. The three most common resistance patterns were ampicillin, dihydrostreptomycin, sulfamethoxypyridazine, tetracycline; ampicillin, dihydrostreptomycin, sulfamethoxypyridazine; dihydrostreptomycin, sulfamethoxypyridazine, tetracycline. Resistance transfer was demonstrated on 267 multiply resistant cultures, of which 181 were able to transfer all or part of their resistance pattern to a drug-sensitive recipient.     

Characteristics of Clostridium botulinum Type F Isolated from the Pacific Coast of the United States

Some of the physiological and biochemical characteristics of a type F strain recently isolated from the United States were studied and compared with those of the prototype Langeland type F strain. The recent isolates were nonproteolytic, fermented sucrose and ribose, produced spores of low thermal resistance, produced a protoxin activated by trypsin, and grew and produced toxin at 38 F (3.3 C) from a spore inoculum. The prototype Langeland strain was proteolytic, did not ferment sucrose or ribose, and produced spores of relatively high thermal resistance, and the toxin of 3-day-old cultures was not activated by trypsin. Approximately two to three times the minimal lethal dose (MLD) of type F toxin from either Langeland or nonproteolytic strains was cross-neutralized by 1,000 anti-MLD of type E antitoxin. Antitoxin serums prepared by immunizing rabbits with the toxoid of the nonproteolytic type F isolate neutralized the toxin of the Langeland strain, but did not show cross-neutralization with the toxins of other types of Clostridium botulinum.     

Salmonella enterica Burden in Harvest-Ready Cattle Populations from the Southern High Plains of the United States

Our objectives were to quantify the Salmonella enterica burdens in harvest-ready cattle and to identify specific at-risk populations of cattle most likely to harbor multiply resistant S. enterica. Hide swabs were collected in abattoirs from three cohorts of cattle (feedlot origin cattle that had achieved desirable harvest characteristics and dairy- and beef-type cows harvested because of poor productivity). Feces were collected from two cohorts housed in feedlots (cattle that had achieved desirable harvest characteristics and animals identified for salvage recovery because of poor productivity). Facilities were visited on four occasions over a 12-month period. Salmonella enterica isolates were recovered, and organisms were quantified using standard microbiological methodologies. Susceptibility to antimicrobial drugs and serotype were determined for one S. enterica isolate per sample. Salmonella enterica was recovered from 55.6% of 1,681 samples. The prevalences on hides and in feces were 69.6% and 30.3%, respectively. The concentrations of S. enterica organisms averaged (as determined by the most probable number technique) 1.82 log₁₀/100 cm² of hides and 0.75 log₁₀/g of feces. None of the isolates recovered from cattle that had achieved desirable harvest characteristics were resistant to four or more drugs. For isolates recovered from animals with poor productivity characteristics, 6.5% were resistant to four or more drugs. Twenty-two serovars were identified, with the most common being Salmonella enterica serovar Anatum (25.5%), Salmonella enterica serovar Montevideo (22.2%), and Salmonella enterica serovar Cerro (12.5%). High-level resistance, i.e., resistance to four or more drugs, was clustered within a few relatively uncommon serovars. These results demonstrate that even though S. enterica isolates are readily recoverable from harvest-ready cattle, multiply resistant variants are rare and are associated with specific serovars in cattle harvested because of poor productivity characteristics.     

Evidence that descendants of three founders constitute about 25% of hemophilia B in the United States

In our sample of 160 consecutive Caucasian hemophiliacs, 14 (9%) had a G----A transition at bp 10,430 that substitutes serine for glycine 60 in the first EGF domain of the factor IX molecule. Each of these hemophiliacs had clinically mild disease. Haplotype data and familial pedigrees indicate that 12 of these hemophiliacs are likely to be related to a common ancestor. The 13th and 14th patients possess different haplotypes and thus represent independent origins of the mutation. In addition, we have screened these 160 hemophiliacs for the previously reported mutations resulting from founder effects at IIe397----Thr and Thr296----Met. Herein we report an additional nine hemophiliacs with the mutant Thr397 allele and five additional hemophiliacs with the mutant Met296 allele. Haplotype data for these 14 hemophiliacs support the original founder effect hypotheses for these mutations. In total, the above three mutations are found in 44 of the 160 seemingly unrelated Caucasian hemophiliacs (28%). The sample includes patients from all regions of the United States and Ontario, Canada. Descendants of these three founders comprise approximately two-thirds of the missense mutations found in our sample of Caucasian hemophiliacs with clinically mild disease.     

Identification and Retting Efficiencies of Fungi Isolated from Dew-Retted Flax in the United States and Europe

Seven strains of filamentous fungi and one yeast were isolated from flax that was dew retted in the United States. These filamentous fungi were subcultured to purity and identified, and six appear not to have been reported earlier as isolates from dew-retted flax. Five of the purified U.S. strains, two fungi isolated from flax that was dew retted in Europe, and a laboratory culture of Aspergillus sojae were tested for their ability to ret flax stems. The monocultures were evaluated for the degree of retting, fiber strength, dry weight loss, and tactile response (i.e., feel of softness) as reflected in the retted fiber. Structural modifications of representative samples of the retted flax were assessed by scanning electron microscopy. All of the filamentous fungi were able to carry out some retting, whereas the isolated yeast could not. All organisms produced pectinases when they were cultivated in shake flasks on ball-milled flax as the sole carbon source. Some fungi also produced cellulases, mannanases, and xylanases. Rhizomucor pusillus and Fusarium lateritium were noteworthy as retting organisms by their high level of pectinase activity, ability to attack noncellulosic cell types without attacking cellulose, capacity to penetrate the cuticular surface of the stem, and efficient fiber release from the core. The results indicated that these organisms deserve further study as potential organisms for retting of bast fibers in industrial applications.