Chen Nadler

The Type III Secretion Effector NleE Inhibits NF-κB Activation

The complex host-pathogen interplay involves the recognition of the pathogen by the hosts innate immune system and countermeasures taken by the pathogen. Detection of invading bacteria by the host leads to rapid activation of the transcription factor NF-kappaB, followed by inflammation and eradication of the intruders. In response, some pathogens, including enteropathogenic Escherichia coli (EPEC), acquired means of blocking NF-kappaB activation. We show that inhibition of NF-kappaB activation by EPEC involves the injection of NleE into the host cell. Importantly, we show that NleE inhibits NF-kappaB activation by preventing activation of IKKbeta and consequently the degradation of the NF-kappaB inhibitor, IkappaB. This NleE activity is enhanced by, but is not dependent on, a second injected effector, NleB. In conclusion, this study describes two effectors, NleB and NleE, with no similarity to other known proteins, used by pathogens to manipulate NF-kappaB signaling pathways.

Metalloprotease type III effectors that specifically cleave JNK and NF-κB.

Two major arms of the inflammatory response are the NF‐κB and c‐Jun N‐terminal kinase (JNK) pathways. Here, we show that enteropathogenic Escherichia coli (EPEC) employs the type III secretion system to target these two signalling arms by injecting host cells with two effector proteins, NleC and NleD. We provide evidence that NleC and NleD are Zn‐dependent endopeptidases that specifically clip and inactivate RelA (p65) and JNK, respectively, thus blocking NF‐κB and AP‐1 activation. We show that NleC and NleD co‐operate and complement other EPEC effectors in accomplishing maximal inhibition of IL‐8 secretion. This is a remarkable example of a pathogen using multiple effectors to manipulate systematically the host inflammatory response signalling network.

Transient Shielding of Intimin and the Type III Secretion System of Enterohemorrhagic and Enteropathogenic Escherichia coli by a Group 4 Capsule

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains represent a major global health problem. Their virulence is mediated by the concerted activity of an array of virulence factors including toxins, a type III protein secretion system (TTSS), pili, and others. We previously showed that EPEC O127 forms a group 4 capsule (G4C), and in this report we show that EHEC O157 also produces a G4C, whose assembly is dependent on the etp, etk, and wzy genes. We further show that at early time points postinfection, these G4Cs appear to mask surface structures including intimin and the TTSS. This masking inhibited the attachment of EPEC and EHEC to tissue-cultured epithelial cells, diminished their capacity to induce the formation of actin pedestals, and attenuated TTSS-mediated protein translocation into host cells. Importantly, we found that Ler, a positive regulator of intimin and TTSS genes, represses the expression of the capsule-related genes, including etp and etk. Thus, the expression of TTSS and G4C is conversely regulated and capsule production is diminished upon TTSS expression. Indeed, at later time points postinfection, the diminishing capsule no longer interferes with the activities of intimin and the TTSS. Notably, by using the rabbit infant model, we found that the EHEC G4C is required for efficient colonization of the rabbit large intestine. Taken together, our results suggest that temporal expression of the capsule, which is coordinated with that of the TTSS, is required for optimal EHEC colonization of the host intestine.

Mutational Analysis of the Locus of Enterocyte Effacement-Encoded Regulator (Ler) of Enteropathogenic Escherichia coli

The locus of enterocyte effacement (LEE) pathogenicity island of enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) comprises a cluster of operons encoding a type III secretion system and related proteins, all of which are essential for bacterial colonization of the host intestines. The LEE1 operon encodes Ler, which positively regulates many EPEC and EHEC virulence genes located in the LEE region and elsewhere in the chromosome. In addition, Ler is a specific autorepressor of LEE1 transcription. To better understand the function of Ler, we screened for Ler mutants defective in autorepression. We isolated 18 different point mutations in Ler, rendering it defective in autorepression and in DNA binding. Among these mutants were those defective in positive regulation as well as in autorepression, dominant-negative mutants, and a mutant deficient in oligomerization. Importantly, a group of Ler autorepression mutants complemented an EPEC ler deletion mutant for transcription activation in a dosage-dependent manner, suggesting that Ler and possibly other autorepressors have an intrinsic compensatory mechanism that enables them to sustain mutations. In addition, the phenotypes of the different mutants identified by the screen define a novel domain in Ler that is required for oligomerization.

Characterization of Enteropathogenic Escherichia coli Mutants That Fail To Disrupt Host Cell Spreading and Attachment to Substratum

ABSTRACT Upon infection of host cells, enteropathogenic Escherichia coli (EPEC) delivers a set of effector proteins into the host cell cytoplasm via the type III secretion system (TTSS). The effectors subvert various host cell functions. We found that EPEC interferes with the spreading and ultimately with the attachment of suspended fibroblasts or epithelial cells, and we isolated mini-Tn10kan insertion mutants that failed to similarly affect host cells. In most mutants, the insertion sites were mapped to genes encoding TTSS components, including cesD, escC, escJ, escV, espD, sepL, espB, and escF. Other mutants contained insertions in micC or upstream of bfpP, yehL, or ydeP. The insertion upstream of ydeP was associated with a reduction in TTSS protein production and was studied further. To determine whether the apparent repression was due to constitutive expression of the downstream encoded genes, ydeP and ydeO expression vectors were constructed. Expression of recombinant YdeP, YdeO, or EvgA, a positive regulator of both ydeP and ydeO, repressed TTSS protein production. Our results suggest that upon activation of the EvgAS two-component system, EvgA (the response regulator) activates both ydeP and ydeO expression and that YdeP and YdeO act conjointly, directly or indirectly repressing expression of the TTSS genes.

Cycling of Etk and Etp Phosphorylation States Is Involved in Formation of Group 4 Capsule by Escherichia coli

Capsules frequently play a key role in bacterial interactions with their environment. Escherichia coli capsules were categorized as groups 1 through 4, each produced by a distinct mechanism. Etk and Etp are members of protein families required for the production of group 1 and group 4 capsules. These members function as a protein tyrosine kinase and protein tyrosine phosphatase, respectively. We show that Etp dephosphorylates Etk in vivo, and mutations rendering Etk or Etp catalytically inactive result in loss of group 4 capsule production, supporting the notion that cyclic phosphorylation and dephosphorylation of Etk is required for capsule formation. Notably, Etp also becomes tyrosine phosphorylated in vivo and catalyzes rapid auto-dephosphorylation. Further analysis identified Tyr121 as the phosphorylated residue of Etp. Etp containing Phe, Glu or Ala in place of Tyr121 retained phosphatase activity and catalyzed dephosphorylation of Etp and Etk. Although EtpY121E and EtpY121A still supported capsule formation, EtpY121F failed to do so. These results suggest that cycles of phosphorylation and dephosphorylation of Etp, as well as Etk, are involved in the formation of group 4 capsule, providing an additional regulatory layer to the complex control of capsule production.

Demographics and referral patterns of a university-based oral maxillofacial radiology clinic over a 20 year period

OBJECTIVE The aim of this study was to review the referral patterns, distribution of interpretations, and type of diagnostic imaging used in a university based oral maxillofacial radiology clinic for patients referred for consultation from both dental and nondental clinicians. STUDY DESIGN The database of the Special Procedures clinic in the oral radiology department of the University of Toronto, Faculty of Dentistry, containing over 5000 entries, from 1993 to 2013, was queried. Using descriptive categorical analysis, the results were analyzed to describe patient demographic characteristics, the specialty of the referring clinicians, the imaging modalities used, and the interpretation provided. RESULTS Most referrals were from oral and maxillofacial surgeons and general dentists. Approximately 25% of referrals were interpreted as variations of normal anatomic structures. The most common reasons for referral were intraosseous lesions (42%), temporomandibular joints (39%), and sialography. Ten percent of all referrals were for recall examinations. The distribution of image modalities has changed through the years covered by this study. CONCLUSIONS This study reflects a lower proportion of referrals reported as normal structures and their variations, than reported in a previous comparable study.

Novel parotid sialo-cone-beam computerized tomography features in patients with suspected Sjogren’s syndrome

OBJECTIVE Sjogrens syndrome (SjS) causes salivary gland impairment leading to oral dryness. Parotid sialo-cone-beam computerized tomography (sialo-CBCT) demonstrates ductal architecture and to a lesser extent gland activity. This study characterizes radiographic features of patients suspected for SjS and looks for a possible correlation with the diagnosis of SjS. METHODS The clinical and radiographic data of suspected SjS/dry mouth patients referred for sialo-CBCT in 2011-2014 were reviewed retrospectively. Two observers studied the scans for various radiographic features including duct morphology, level of branching, ductopenia and sialectasia. These features were analysed taking the specific clinical data and two sets of SjS criteria: The 2002 American-European Consensus Group (AECG) and the 2012 American College of Rheumatology (ACR) Group. RESULTS Sialo-CBCT scans of 67-referred patients suffering from dry mouth (115 parotid glands) were included. Intraradiographic association was found between ductopenia and all other radiographic parameters. Minimal, yet important, radiographic differences were found between left and right parotid glands. AECG-confirmed-SjS patients showed strong correlation with radiographic features, whereas ACR 2012-confirmed-SjS patients did not. CONCLUSION Sialo-CBCT demonstrates novel radiographic features which may clarify the diagnosis of SjS. Further studies are needed to determine the role of sialo-CBCT in diagnosis of SjS.