After >8,000 infections and >700 deaths worldwide, the pathogenesis of the new infectious disease, severe acute respiratory syndrome (SARS), remains poorly understood. We investigated 18 autopsies of patients who had suspected SARS; 8 cases were confirmed as SARS. We evaluated white blood cells from 22 confirmed SARS patients at various stages of the disease. T lymphocyte counts in 65 confirmed and 35 misdiagnosed SARS cases also were analyzed retrospectively. SARS viral particles and genomic sequence were detected in a large number of circulating lymphocytes, monocytes, and lymphoid tissues, as well as in the epithelial cells of the respiratory tract, the mucosa of the intestine, the epithelium of the renal distal tubules, the neurons of the brain, and macrophages in different organs. SARS virus seemed to be capable of infecting multiple cell types in several organs; immune cells and pulmonary epithelium were identified as the main sites of injury. A comprehensive theory of pathogenesis is proposed for SARS with immune and lung damage as key features.

Biofilm formation by the periodontal pathogen Aggregatibacter actinomycetemcomitans is dependent upon autoinducer-2 (AI-2)-mediated quorum sensing. However, the components that link the detection of the AI-2 signal to downstream gene expression have not been determined. One potential regulator is the QseBC two-component system, which is part of the AI-2-dependent response pathway that controls biofilm formation in Escherichia coli. Here we show that the expression of QseBC in A. actinomycetemcomitans is induced by AI-2 and that induction requires the AI-2 receptors, LsrB and/or RbsB. Additionally, inactivation of qseC resulted in reduced biofilm growth. Since the ability to grow in biofilms is essential for A. actinomycetemcomitans virulence, strains that were deficient in QseC or the AI-2 receptors were examined in an in vivo mouse model of periodontitis. The ⌬qseC mutant induced significantly less alveolar bone resorption than the wild-type strain (P < 0.02). Bone loss in animals infected with the ⌬qseC strain was similar to that in sham-infected animals. The ⌬lsrB, ⌬rbsB, and ⌬lsrB ⌬rbsB strains also induced significantly less alveolar bone resorption than the wild type (P < 0.03, P < 0.02, and P < 0.01, respectively). However, bone loss induced by a ⌬luxS strain was indistinguishable from that induced by the wild type, suggesting that AI-2 produced by indigenous microflora in the murine oral cavity may complement the ⌬luxS mutation. Together, these results suggest that the QseBC two-component system is part of the AI-2 regulon and may link the detection of AI-2 to the regulation of downstream cellular processes that are involved in biofilm formation and virulence of A. actinomycetemcomitans.

Autoinducer 2 (AI-2) is required for the growth of Aggregatibacter (Actinobacillus) actinomycetemcomitans in culture under conditions of iron limitation. However, in vivo this organism thrives in a complex multispecies biofilm that forms in the human oral cavity. In this report, we show that adherent growth of A. actinomycetemcomitans on a saliva-coated surface, but not planktonic growth under iron-replete conditions, is defective in a LuxS-deficient background. Biofilm growth of the luxS mutant exhibited lower total biomass and lower biofilm depth than those for the wild-type strain. Normal biofilm growth of the luxS mutant was restored genetically by introduction of a functional copy of luxS and biochemically by addition of partially purified AI-2. Furthermore, introduction of S-adenosylhomocysteine hydrolase, which restores the metabolism of S-adenosylmethionine in the absence of LuxS, into A. actinomycetemcomitans did not complement the luxS mutation unless AI-2 was added in trans. This suggests that AI-2 itself is required for biofilm growth by A. actinomycetemcomitans. A biofilm growth deficiency similar to that of the LuxS-deficient strain was also observed when a gene encoding the AI-2-interacting protein RbsB or LsrB was inactivated. Biofilm formation by A. actinomycetemcomitans was virtually eliminated upon inactivation of both rbsB and lsrB. In addition, biofilm growth by wild-type A. actinomycetemcomitans was reduced in the presence of ribose, which competes with AI-2 for binding to RbsB. These results suggest that RbsB and LsrB function as AI-2 receptors in A. actinomycetemcomitans and that the development of A. actinomycetemcomitans biofilms requires AI-2.

In addition to the lungs, the gastrointestinal tract is another target of SARS-CoV infection, as the intestinal epithelial cells and mucosal lymphoid tissue are infected. The findings provide possible explanations for the gastrointestinal symptoms and the presence of virus in the stool of SARS patients.

Autoinducer 2 (AI-2) produced by the oral pathogen Actinobacillus actinomycetemcomitans influences growth of the organism under iron limitation and regulates the expression of iron uptake genes. However, the cellular components that mediate the response of A. actinomycetemcomitans to AI-2 have not been fully characterized. Analysis of the complete genome sequence of A. actinomycetemcomitans (www.oralgen.lanl.gov) indicated that the RbsB protein was related to LuxP, the AI-2 receptor of Vibrio harveyi. To determine if RbsB interacts with AI-2, the bioluminescence of the reporter strain V. harveyi BB170 (sensor 1؊, sensor 2؉) was determined after stimulation with partially purified AI-2 from A. actinomycetemcomitans or conditioned medium from V. harveyi cultures in the presence and absence of purified six-His-tagged RbsB. RbsB efficiently inhibited V. harveyi bioluminescence induced by both A. actinomycetemcomitans AI-2 and V. harveyi AI-2 in a dose-dependent manner, suggesting that RbsB competes with LuxP for AI-2. Fifty percent inhibition occurred with approximately 0.3 nM RbsB for A. actinomycetemcomitans AI-2 and 15 nM RbsB for V. harveyi AI-2. RbsB-mediated inhibition of V. harveyi bioluminescence was reversed by the addition of 50 mM ribose, suggesting that A. actinomycetemcomitans AI-2 and ribose bind at the same site of RbsB. The RbsB/AI-2 complex was thermostable since A. actinomycetemcomitans AI-2 could not be recovered by heating. This was not due to heat inactivation of A. actinomycetemcomitans AI-2 since signal activity was unaffected by heating in the absence of RbsB. Furthermore, an isogenic A. actinomycetemcomitans mutant that was unable to express rbsB was deficient in depleting A. actinomycetemcomitans AI-2 from solution relative to the wild-type organism. Inactivation of rbsB also influenced the ability of the organism to grow under iron-limiting conditions. The mutant strain attained a cell density of approximately 30% that of the wild-type organism under iron limitation. In addition, real-time PCR showed that the expression of afuABC, encoding a major ferric ion transporter, was reduced by approximately eightfold in the rbsB mutant. This phenotype was similar to that of a LuxS-deficient mutant of A. actinomycetemcomitans that is unable to produce AI-2. Together, our results suggest that RbsB may play a role in the response of A. actinomycetemcomitans to AI-2.

Our previous studies showed that the Aggregatibacter actinomycetemcomitans RbsB protein interacts with cognate and heterologous autoinducer 2 (AI-2) signals and suggested that the rbsDABCK operon encodes a transporter that may internalize AI-2 (D. James et al., Infect. Immun. 74:4021-4029, 2006.). However, A. actinomycetemcomitans also possesses genes related to the lsr operon of Salmonella enterica serovar Typhimurium which function to import AI-2. Here, we show that A. actinomycetemcomitans LsrB protein competitively inhibits the interaction of the Vibrio harveyi AI-2 receptor (LuxP) with AI-2 from either A. actinomycetemcomitans or V. harveyi. Interestingly, LsrB was a more potent inhibitor of LuxP interaction with AI-2 from V. harveyi whereas RbsB competed more effectively with LuxP for A. actinomycetemcomitans AI-2. Inactivation of lsrB in wild-type A. actinomycetemcomitans or in an isogenic RbsB-deficient strain reduced the rate by which intact bacteria depleted A. actinomycetemcomitans AI-2 from solution. Consistent with the results from the LuxP competition experiments, the LsrB-deficient strain depleted AI-2 to a lesser extent than the RbsB-deficient organism. Inactivation of both lsrB and rbsB virtually eliminated the ability of the organism to remove AI-2 from the extracellular environment. These results suggest that A. actinomycetemcomitans possesses two proteins that differentially interact with AI-2 and may function to inactivate or facilitate internalization of AI-2.Autoinducer 2 (AI-2) is a quorum-sensing signal that was initially identified in Vibrio harveyi (2) and is produced by the luxS gene (28, 29). AI-2 is produced by LuxS-catalyzed cleavage of S-ribosylhomocysteine to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione (36), which in turn undergoes further rearrangement to produce AI-2. However, two distinct structural forms of AI-2 have been identified. Salmonella enterica serovar Typhimurium produces 2R,4S-2,3,3,4-methyltetrahydroxytetrahydrofuran (R-THMF) whereas Vibrio subsp. produce the borate diester form of S-THMF (6, 22). AI-2-dependent quorum sensing is quite complex and is involved in the regulation of a variety of genes in Vibrio species, including the lux operon of V. harveyi. Detection of AI-2 by V. harveyi is mediated by LuxP, a periplasmic AI-2 receptor (6) that associates with the LuxQ sensor kinase-phosphatase (23) and initiates a phosphotransfer cascade involving LuxU (11) and the response regulator LuxO (10). LuxO in turn influences the expression of multiple small regulatory RNAs (Qrr regulators) that influence the expression of LuxR (15, 16), the master regulator of the lux operon (16). Recently, an additional twocomponent system encoded by varSA in Vibrio cholerae has been shown to converge on the quorum-sensing circuit by regulating the expression of the small RNAs encoded by csrBCD which control the expression of Qrr via CsrA (15).LuxS is highly conserved in a wide range of gram-positive and gram-negative bacteria, and many, if not all, of these organisms produ...

Background Cortical inhibition plays a critical role in controlling and modulating cortical excitation and a more detailed understanding of the neuronal circuits contributing to each will provide more insight into their roles in complex cortical computations. Traditional neuronal tracers lack a means for easily distinguishing between circuits of inhibitory and excitatory neurons. To overcome this limitation, we developed a technique for retrogradely labeling inputs to local clusters of inhibitory or excitatory neurons, but not both, using neurotropic adeno-associated and lentiviral vectors, cell-type specific promoters and a modified rabies virus. Results Applied to primary visual cortex (V1) in mouse, the cell-type specific tracing technique labeled thousands of presynaptically connected neurons, and revealed that the dominant source of input to inhibitory and excitatory neurons is local in origin. Neurons in other visual areas are also labeled; the percentage of these inter-cortical inputs to excitatory neurons is somewhat higher (~20%) than to inhibitory neurons (<10%), suggesting that inter-cortical connections have less direct control over inhibition. The inputs to inhibitory neurons were also traced in cat V1, and when aligned with the orientation preference map, revealed for the first time that long-range inputs to inhibitory neurons are well tuned to orientation. Conclusions These novel findings for inhibitory and excitatory circuits in the visual cortex demonstrate the efficacy of our new technique and its ability to work across species, including larger-brained mammals such as the cat. This paves the way for better understanding the roles of specific cell-types in higher-order perceptual and cognitive processes.

Comparable NF-κB activation and p38 phosphorylation in TNF-treated wild-type and Actin mut cells also indicates that reduced expression of actin only selectively blocked some of the TNF-induced cellular changes. Actin cleavage involved in apoptosis does not occur in TNF-treated L929 cell death, as in HeLa cells. Consistent over-expression of a caspase-cleaved product, a 15 kDa actin fragment, had no effect on TNF-induced necrosis of L929 cell. By contrast, TNF-induced mitochondria clustering and ROS production were dramatically reduced in Actin mut cells, indicating that actin-deficiency-mediated TNF resistance is most likely due to impaired mitochondrial responses to TNF stimulation. Our findings suggest that a full complement of actin is required for transduction of a cell death signal to mitochondria in TNF-treated L929 cells.