Tyler G. Kimbrough

Structural characterization of the molecular platform for type III secretion system assembly

Type III secretion systems (TTSSs) are multi-protein macromolecular ‘machines’ that have a central function in the virulence of many Gram-negative pathogens by directly mediating the secretion and translocation of bacterial proteins (termed effectors) into the cytoplasm of eukaryotic cells. Most of the 20 unique structural components constituting this secretion apparatus are highly conserved among animal and plant pathogens and are also evolutionarily related to proteins in the flagellar-specific export system. Recent electron microscopy experiments have revealed the gross ‘needle-shaped’ morphology of the TTSS, yet a detailed understanding of the structural characteristics and organization of these protein components within the bacterial membranes is lacking. Here we report the 1.8-Å crystal structure of EscJ from enteropathogenic Escherichia coli (EPEC), a member of the YscJ/PrgK family whose oligomerization represents one of the earliest events in TTSS assembly. Crystal packing analysis and molecular modelling indicate that EscJ could form a large 24-subunit ‘ring’ superstructure with extensive grooves, ridges and electrostatic features. Electron microscopy, labelling and mass spectrometry studies on the orthologous Salmonella typhimurium PrgK within the context of the assembled TTSS support the stoichiometry, membrane association and surface accessibility of the modelled ring. We propose that the YscJ/PrgK protein family functions as an essential molecular platform for TTSS assembly.

Assembly of the type III secretion needle complex of Salmonella typhimurium.

The type III secretion needle complex (NC) of Salmonella typhimurium is a complex secretory system that functions to translocate virulence proteins into eukaryotic cells. Evolutionarily it is related to bacterial flagella. Assembly of the NC occurs through ordered secretion, polymerization, and assembly, and requires the coordinated expression and association of over 20 different proteins. Recent progress in the understanding of the assembly and architecture of the NC is reviewed.

SspA Is Required for Lethal Salmonella enterica Serovar Typhimurium Infections in Calves but Is Not Essential for Diarrhea

ABSTRACT Salmonella pathogenicity island 1 (SPI-1) encodes virulence determinants, which are important for enteropathogenicity in calves. To determine whether the Salmonella entericaserovar Typhimurium SPI-1 effector proteins SspA and SptP are important for enteropathogenicity, strains lacking these proteins were tested during oral infection of calves. Calves infected with asptP mutant or its isogenic parent developed diarrhea and lethal morbidity. In contrast, calves infected with an sspAmutant developed diarrhea, which resolved within 10 days but did not result in mortality. The sspA mutant was recovered from bovine intestinal tissues at numbers similar to those obtained for its isogenic parent and caused marked intestinal lesions. Thus, the severity of pathological changes caused by serovar Typhimurium strains or their ability to cause diarrhea were not predictive of their ability to cause lethal morbidity in calves. We conclude that factors other than or in addition to bacterial colonization, intestinal lesions, or electrolyte loss contribute to lethal morbidity in calves infected with serovar Typhimurium.

Symptom duration and CT findings in pediatric deep neck infection

Objective: To investigate whether children with less than 48 hours of localized symptoms of deep neck infection are less likely to have an abscess on CT scan. Study Design: Case series. Subjects and Methods: The charts of children seen in a tertiary childrens hospital for deep neck infections between 2000 and 2007 were reviewed. Results: Of 179 children identified, 167 (93.3%) underwent a CT scan of the neck of which 102 (61.1%) were positive for abscess. There was no significant difference in the rate of abscess on CT between children with less than 48 hours of localizing symptoms and 48 or more hours of symptoms at 58.1 percent and 58.3 percent, respectively (P = 0.98). Furthermore, there was no significant difference in age, gender, C-reactive protein levels, disease location, or length of stay between children with and without abscess on CT. White blood cell counts were significantly higher in the abscess group (P = 0.01); however, the median white blood cell count in both groups was above normal. Conclusion: Because duration of symptoms does not predict finding of abscess on CT, it is appropriate to obtain a CT scan upon presentation in all children with symptoms concerning for neck abscess.

S256 – Microbiology of Pediatric Head and Neck Abscesses:

Objectives To report the microbiology of pediatric head and neck abscesses. Methods The records of a tertiary pediatric hospital were searched and all children with infections of the head and neck region between 2000 and 2007 were identified. Cases of peritonsillar abscess were excluded. Data regarding presentation, physical exam findings, radiographic findings, and treatment were extracted. All children who were diagnosed with abscesses and treated surgically were further identified, and the results of intraoperative cultures were reviewed. Results A total of 179 children with infections of the head and neck were treated between the years of 2000 and 2007 of which 57% (102 children) were diagnosed with abscesses. 71% (72 children) with abscesses were treated surgically. Of 62 children with drainable abscesses, culture data was available for 54 children. A single organism was isolated in 24.1% of cultures, 2 organisms in 18.5% of cultures and 3 organisms in 16.7% of cultures. More than 3 organisms were identified in 38.9% of cultures. The most commonly isolated organisms included alpha-hemolytic streptococci (57.4%), group A streptococci (37.0%), anaerobes (29.6%), hemophilus (22.2%), neisseria (22.2%), and “respiratory flora” (20.4%). Other streptococcal species (14.8%), candida (14.8%), staphylococci (11.1%), stomatococci (11.1%) and other organisms (7.4%) were also identified. One culture showed no growth. No cases of methicillin-resistant staphylococcus aureus (MRSA) were encountered. Conclusions In our series, streptococci and anaerobic bacteria were the most commonly isolated organisms, and almost 40% of cultures identified greater than 3 different organisms. No cases of MRSA were encountered.

S253 – Management of Pediatric Skull Base Abscesses

Objectives To describe the presentation and management of pediatric deep neck space infections located at the skull base. Methods Design: Retrospective review. Setting: Tertiary childrens hospital. The study population comprised pediatric patients admitted to the hospital for management of deep neck space infections between 2000 and 2007. Main Outcome Measure: Resolution of abscess. Results Over the study period, 179 patients were admitted for management of deep neck space infections. Of these, 10 (6%) were localized to the skull base by CT scan. 7 of the 10 met radiographic criteria for an abscess; the remaining 3 were designated edema or phlegmon. Patients ranged in age from 2 to 8 years old. The most common presenting symptoms were sore throat (60%), stiff or sore neck (50%), neck or facial swelling (20%), and drooling (20%). Otitis media and Streptococcal pharyngitis were the most common co-morbidities found in 40% of patients. Initial treatment consisted of IV antibiotics in all cases. 70% resolved with medical management alone. 3 patients failed to resolve following 48 hrs of IV antibiotics and were taken to the operating room for abscess drainage, which was unsuccessful in all cases. Repeat CT after an additional 48 hours of medical therapy indicated persistent abscess in 2 of the 3 operative cases. Repeat drainage was successful in only 1 of these. Conclusions Skull base abscesses in children can be effectively managed in most cases by IV antibiotics alone. Surgical drainage is difficult and often unsuccessful and therefore poses unnecessary risk to the patient.

R043: HOP, A Novel Tumor Suppressor Gene in Head and Neck SCCA

PROBLEM: Despite recent advances, five-year survival rates for head and neck cancers have not improved significantly over the past 20 years. Therefore, there is a critical need to establish more efficacious diagnositics and therapies. Studies using Gene Chip microarray analysis which identified a list of 2,890 genes differentially expressed between HNSCCA tumors and normal mucosa have been previously reported. Expression of the gene encoding the Homeo-domain-only protein (HOP) was greatly decreased in all tumor samples. This finding was supported by work from other groups which suggested that HOP may function as a potent tumor suppressor. METHODS: DNA sequencing was performed on selected patient tumor samples to examine the integrity of the HOP locus. RT PCR and gene fusion experiments were used to study the expression of HOP in different HNSCC cell lines and keratinocyte (noncarcinoma) cell lines. The effect of constitutive activation of the NFKB pathway on HOP expression was also examined. RESULTS: Sequencing of the HOP locus revealed that loss of HOP expression in patient tumor samples and HNSCC cell lines was not the result of genetic mutation. RT PCR and promoter fusion studies confirmed the loss of HOP expression in the HNSCC cells whereas the noncarcinoma keratinocyte cell lines maintained high levels of HOP expression. Activation of the inflammatory pathway through constitutive NFKB expression did not alter the level of HOP expression. CONCLUSION: Loss of HOP expression in HNSCC is not the result of genetic mutation, suggesting the loss of upstream activators or other epigenetic mechanisms. While inflammation via NFKB pathway activation has been implicated in HNSCC pathogenesis, it does not appear to regulate HOP expression. SIGNIFICANCE: Further insight into the underlying molecular pathophysiology of HNSCC may lead to the discovery of unique biomarkers and novel molecular therapeutic targets. SUPPORT: Lions Hearing Foundation Research Grant, University of Minnesota.