No abstract

f Hypervirulent (hypermucoviscous) Klebsiella pneumoniae (hvKP) strains are an emerging variant of "classical" K. pneumoniae (cKP) that cause organ and life-threatening infection in healthy individuals. An understanding of hvKP-specific virulence mechanisms that enabled evolution from cKP is limited. Observations by our group and previously published molecular epidemiologic data led us to hypothesize that hvKP strains produced more siderophores than cKP strains and that this trait enhanced hvKP virulence. Quantitative analysis of 12 hvKP strains in iron-poor minimal medium or human ascites fluid showed a significant and distinguishing 6-to 10-fold increase in siderophore production compared to that for 14 cKP strains. Surprisingly, highpressure liquid chromatography (HPLC)-mass spectrometry and characterization of the hvKP strains hvKP1, A1142, and A1365 and their isogenic aerobactin-deficient (⌬iucA) derivatives established that aerobactin accounted for the overwhelming majority of increased siderophore production and that this was not due to gene copy number. Further, aerobactin was the primary factor in conditioned medium that enhanced the growth/survival of hvKP1 in human ascites fluid. Importantly the ex vivo growth/ survival of hvKP1 ⌬iucA was significantly less than that of hvKP1 in human ascites fluid, and the survival of outbred CD1 mice challenged subcutaneously or intraperitoneally with hvKP1 was significantly less than that of mice challenged with hvKP1 ⌬iucA. The lowest subcutaneous and intraperitoneal challenge inocula of 3 ؋ 10 2 and 3.2 ؋ 10 1 CFU, respectively, resulted in 100% mortality, demonstrating the virulence of hvKP1 and its ability to cause infection at a low dose. These data strongly support that aerobactin accounts for increased siderophore production in hvKP compared to cKP (a potential defining trait) and is an important virulence factor.

Summary Protein synthesis is crucial for the maintenance of long-term-memory-related synaptic plasticity. The prion-like cytoplasmic polyadenylation element-binding protein 3 (CPEB3) regulates the translation of several mRNAs important for long-term synaptic plasticity in the hippocampus. Here, we provide evidence that the prion-like aggregation and activity of CPEB3 is controlled by SUMOylation. In the basal state, CPEB3 is a repressor and is soluble. Under these circumstances, CPEB3 is SUMOylated in hippocampal neurons both in vitro and in vivo. Following neuronal stimulation, CPEB3 is converted into an active form that promotes the translation of target mRNAs, and this is associated with a decrease of SUMOylation and an increase of aggregation. A chimeric CPEB3 protein fused to SUMO cannot form aggregates and cannot activate the translation of target mRNAs. These findings suggest a model whereby SUMO regulates translation of mRNAs and structural synaptic plasticity by modulating the aggregation of the prion-like protein CPEB3.

No abstract

The heat-labile enterotoxins (HLT) of Vibrio cholerae and Escherichia coli constitute a family of bacterial toxins that are related in structure and function (10,11,16,35). Both are oligomeric protein toxins composed of one A polypeptide and five B polypeptides in which the quaternary structure is maintained by noncovalent bonds between the A polypeptide and a pentameric ring of B subunits (7,13,32). The biological effects of the enterotoxins are determined by the binding specificity of the fully assembled B subunits and the enzymatic activity of the A subunit. The pentameric ring formed by the B subunits mediates binding to the sugar residues of gangliosides present on the surface of various eukaryotic cells (3,18).Two serogroups of HLT have been distinguished on the basis of distinct immunoreactivity (15, 28). Serogroup I consists of cholera toxin (CT) and the E. coli HLT LT-I, which includes two antigenic variants isolated from humans and pigs, designated respectively (19,28). Serogroup II enterotoxins include E. coli type II HLT initially designated LT-like toxins and later called LT-II enterotoxins (9). Based on immunoreactivity and amino acid sequence homology, two antigenic variants of LT-II, designated LT-IIa and LT-IIb, have been isolated (9)(10)(11)17). Although serogroup I and serogroup II enterotoxins induce similar morphological effects on Y1 adrenal cells and activate adenylate cyclase in cell cultures, both LT-IIa and LT-IIb appear to be more potent than either CT or LT-I in Y1 adrenal cell assays; however, neither LT-IIa nor LT-IIb induces the typical fluid accumulation in ligated ileal loops observed with CT and LT-I (16). In human T84 intestinal cells, only CT elicited a cyclic AMP-dependent chloride response that is responsible for the massive effusion of water into the lumen of the gut (39).Comparison of the predicted amino acid sequences of type I and type II enterotoxins reveals a large degree of variability. While the B polypeptides of CT and LT-I exhibit over 80% homology to each other, both CT and LT-I have less than 14% amino acid sequence homology to the B subunits of either LT-IIa or [28][29][30]. The extensive diversity in amino acid sequences between type I and type II HLT not only results in antigenically distinct groups but also imparts different ganglioside binding specificity to the respective B subunits. Specifically, the high-affinity receptor for CT and LT

Efficient utilization of heme as an iron (Fe) source by Bordetella avium requires bhuR, an Fe-regulated gene which encodes an outer membrane heme receptor. Upstream of bhuR is a 507-bp open reading frame, hereby designated rhuI (for regulator of heme uptake), which codes for a 19-kDa polypeptide. Whereas the 19-kDa polypeptide had homology to a subfamily of alternative sigma factors known as the extracytoplasmic function (ECF) sigma factors, it was hypothesized that rhuI encoded a potential in-trans regulator of the heme receptor gene in trans. Support for the model was strengthened by the identification of nucleotide sequences common to ECF sigma-dependent promoters in the region immediately upstream of bhuR. Experimental evidence for the regulatory activities of rhuI was first revealed by recombinant experiments in which overproduction of rhuI was correlated with a dramatically increased expression of BhuR. A putative rhuI-dependent bhuR promoter was identified in the 199-bp region located proximal to bhuR. When a transcriptional fusion of the 199-bp region and a promoterless lacZ gene was introduced into Escherichia coli, promoter activity was evident, but only when rhuI was coexpressed in the cell. Sigma competition experiments in E. coli demonstrated that rhuI conferred biological properties on the cell that were consistent with RhuI having sigma factor activity. Heme, hemoglobin, and several other heme-containing proteins were shown to be the extracellular inducers of the rhuI-dependent regulatory system. Fur titration assays indicated that expression of rhuI was probably Fur dependent.

Iron (Fe) is an essential element for most organisms which must be obtained from the local environment. In the case of pathogenic bacteria, this fundamental element must be acquired from the fluids and tissues of the infected host. A variety of systems have evolved in bacteria for efficient acquisition of host-bound Fe. The gram-negative bacterium Bordetella avium, upon colonization of the avian upper respiratory tract, produces a disease in birds that has striking similarity to whooping cough, a disease caused by the obligate human pathogen Bordetella pertussis. We describe a B. avium Fe utilization locus comprised of bhuR and six accessory genes (rhuIR and bhuSTUV). Genetic manipulations of B. avium confirmed that bhuR, which encodes a putative outer membrane heme receptor, mediates efficient acquisition of Fe from hemin and hemoproteins (hemoglobin, myoglobin, and catalase). BhuR contains motifs which are common to bacterial heme receptors, including a consensus FRAP domain, an NPNL domain, and two TonB boxes. An N-terminal 32-amino-acid segment, putatively required for rhuIR-dependent regulated expression of bhuR, is present in BhuR but not in other bacterial heme receptors. Two forms of BhuR were observed in the outer membrane of B. avium: a 91-kDa polypeptide consistent in size with the predicted mature protein and a smaller 82-kDa polypeptide which lacks the 104 amino acids found at the N terminus of the 91-kDa form. A mutation in hemA was engineered in B. avium to demonstrate that the bacterium transports heme into the cytoplasm in a BhuR-dependent manner. The role of BhuR in virulence was established in turkey poults by use of a competitive-infection model.

No abstract