Lipooligosaccharides (LOS) of Neisseria gonorrhoeae and Neisseria meningitidis have components that are immunochemically similar to precursors of human blood group antigens. Carbohydrate sequence specificity of the mouse monoclonal antibodies that recognize crossreacting antigens on LOS and human erythrocytes.

We have used mouse mAbs, 3F11 and 06B4, that are specific for highly conserved epitopes of Neisseria gonorrhoeae lipooligosaccharides (LOS) to identify immunochemically similar structures on human erythrocytes. mAb 3F11 agglutinated erythrocytes from all randomly selected adult humans, while mAb 06B4 agglutinated only 80% of the same specimens. The antibodies had an activity with erythrocytes similar to human cold agglutinins in that agglutination occurred at 4 degrees C and decreased with increasing incubation temperature. Human infant erythrocytes were agglutinated less well, but enzymatic treatment of either infant or adult cells resulted in an increase in expression of the 3F11- and 06B4-defined epitopes. Both antibodies bound to a series of neutral glycosphingolipids from human erythrocytes and neutrophils that have a type 2 (Gal beta 1----4GlcNAc) or N-acetyllactosamine structure. Neither antibody bound to glycosphingolipids from human meconium, which have a type 1 (Gal beta 1----3GlcNAc) structure. The antibodies were unable to bind to N-acetyl-lactosamine glycosphingolipids with a nonreducing terminal sialic acid or a Gala1----3Gal disaccharide. Antibody binding also was blocked by the presence of fucose linked to the penultimate glucosamine residue of N-acetyllactosamine glycosphingolipids. Although both antibodies bound to linear and branched-chain N-acetyllactosamine glycosphingolipids, 3F11 had a higher affinity for branched structures than did 06B4. The activity of 3F11 with human adult and infant treated and untreated erythrocytes with N-acetyllactosamine glycosphingolipids, and with LOS was very similar, if not identical, in specificity to 1B2, an mAb prepared from mice inoculated with a linear N-acetyllactosamine glycosphingolipid

Escherichia coli O157:H7 in Feral Swine near Spinach Fields and Cattle, Central California Coast1

We investigated involvement of feral swine in contamination of agricultural fields and surface waterways with Escherichia coli O157:H7 after a nationwide outbreak traced to bagged spinach from California. Isolates from feral swine, cattle, surface water, sediment, and soil at 1 ranch were matched to the outbreak strain

Differentiation of Gram-Negative Bacterial Aerosol Exposure Using Detected Markers in Bronchial-Alveolar Lavage Fluid

The identification of biosignatures of aerosol exposure to pathogens has the potential to provide useful diagnostic information. In particular, markers of exposure to different types of respiratory pathogens may yield diverse sets of markers that can be used to differentiate exposure. We examine a mouse model of aerosol exposure to known Gram negative bacterial pathogens, Francisella tularensis novicida and Pseudomonas aeruginosa. Mice were subjected to either a pathogen or control exposure and bronchial alveolar lavage fluid (BALF) was collected at four and twenty four hours post exposure. Small protein and peptide markers within the BALF were detected by matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and analyzed using both exploratory and predictive data analysis methods; principle component analysis and degree of association. The markers detected were successfully used to accurately identify the four hour exposed samples from the control samples. This report demonstrates the potential for small protein and peptide marker profiles to identify aerosol exposure in a short post-exposure time frame

The EHEC Type III Effector NleL Is an E3 Ubiquitin Ligase That Modulates Pedestal Formation

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome in humans. EHEC colonize the intestinal mucosa and promote the formation of actin-rich pedestals via translocated type III effectors. Two EHEC type III secreted effectors, Tir and EspFu/TccP, are key players for pedestal formation. We discovered that an EHEC effector protein called Non-LEE-encoded Ligase (NleL) is an E3 ubiquitin ligase. In vitro, we showed that the NleL C753 residue is critical for its E3 ligase activity. Functionally, we demonstrated that NleL E3 ubiquitin ligase activity is involved in modulating Tir-mediated pedestal formation. Surprisingly, EHEC mutant strain deficient in the E3 ligase activity induced more pedestals than the wild-type strain. The canonical EPEC strain E2348/69 normally lacks the nleL gene, and the ectopic expression of the wild-type EHEC nleL, but not the catalytically-deficient nleL(C753A) mutant, in this strain resulted in fewer actin-rich pedestals. Furthermore, we showed that the C. rodentium NleL homolog is a E3 ubiquitin ligase and is required for efficient infection of murine colonic epithelial cells in vivo. In summary, our study demonstrated that EHEC utilizes NleL E3 ubiquitin ligase activity to modulate Tir-mediated pedestal formation.National Institutes of Health (U.S.) (grant AI078092)National Institutes of Health (U.S.) (grant AI068655

The Meningococcal Vaccine Candidate Neisserial Surface Protein A (NspA) Binds to Factor H and Enhances Meningococcal Resistance to Complement

Complement forms an important arm of innate immunity against invasive meningococcal infections. Binding of the alternative complement pathway inhibitor factor H (fH) to fH-binding protein (fHbp) is one mechanism meningococci employ to limit complement activation on the bacterial surface. fHbp is a leading vaccine candidate against group B Neisseria meningitidis. Novel mechanisms that meningococci employ to bind fH could undermine the efficacy of fHbp-based vaccines. We observed that fHbp deletion mutants of some meningococcal strains showed residual fH binding suggesting the presence of a second receptor for fH. Ligand overlay immunoblotting using membrane fractions from one such strain showed that fH bound to a ∼17 kD protein, identified by MALDI-TOF analysis as Neisserial surface protein A (NspA), a meningococcal vaccine candidate whose function has not been defined. Deleting nspA, in the background of fHbp deletion mutants, abrogated fH binding and mAbs against NspA blocked fH binding, confirming NspA as a fH binding molecule on intact bacteria. NspA expression levels vary among strains and expression correlated with the level of fH binding; over-expressing NspA enhanced fH binding to bacteria. Progressive truncation of the heptose (Hep) I chain of lipooligosaccharide (LOS), or sialylation of lacto-N-neotetraose LOS both increased fH binding to NspA-expressing meningococci, while expression of capsule reduced fH binding to the strains tested. Similar to fHbp, binding of NspA to fH was human-specific and occurred through fH domains 6–7. Consistent with its ability to bind fH, deleting NspA increased C3 deposition and resulted in increased complement-dependent killing. Collectively, these data identify a key complement evasion mechanism with important implications for ongoing efforts to develop meningococcal vaccines that employ fHbp as one of its components

Comparative Genomic Analysis of Clinical Strains of Campylobacter jejuni from South Africa

BACKGROUND: Campylobacter jejuni is a common cause of acute gastroenteritis and is also associated with the post-infectious neuropathies, Guillain-Barré and Miller Fisher syndromes. In the Cape Town area of South Africa, C. jejuni strains with Penner heat-stable (HS) serotype HS:41 have been observed to be overrepresented among cases of Guillain-Barré syndrome. The present study examined the genetic content of a collection of 32 South African C. jejuni strains with different serotypes, including 13 HS:41 strains, that were recovered from patients with enteritis, Guillain-Barré or Miller Fisher syndromes. The sequence-based typing methods, multilocus sequence typing and DNA microarrays, were employed to potentially identify distinguishing features within the genomes of these C. jejuni strains with various disease outcomes. METHODOLOGY/PRINCIPAL FINDINGS: Comparative genomic analyses demonstrated that the HS:41 South African strains were clearly distinct from the other South African strains. Further DNA microarray analysis demonstrated that the HS:41 strains from South African patients with the Guillain-Barré syndrome or enteritis were highly similar in gene content. Interestingly, the South African HS:41 strains were distinct in gene content when compared to HS:41 strains from other geographical locations due to the presence of genomic islands, referred to as Campylobacter jejuni integrated elements (CJIEs). Only the integrated element CJIE1, a Campylobacter Mu-like prophage, was present in the South African HS:41 strains whereas this element was absent in two closely-related HS:41 strains from Mexico. A more distantly-related HS:41 strain from Canada possessed both integrated elements CJIE1 and CJIE2. CONCLUSION/SIGNIFICANCE: These findings demonstrate that CJIEs may contribute to the differentiation of closely-related C. jejuni strains. In addition, the presence of bacteriophage-related genes in CJIE1 may contribute to the genomic diversity of C. jejuni strains. This comparative genomic analysis of C. jejuni provides fundamental information that potentially could lead to improved methods for analyzing the epidemiology of disease outbreaks