Certain bacterial two-component sensor kinases possess a histidine-containing phosphotransfer (Hpt) domain to carry out a multistep phosphotransferring reaction to a cognate response regulator. Pseudomonas aeruginosa PAO1 contains three genes that encode proteins with an Hpt domain but lack a kinase domain. To identify the sensor kinase coupled to these Hpt proteins, a phosphorelay profiling assay was performed. Among the 12 recombinant orphan sensor kinases tested, 4 of these sensors (PA1611, PA1976, PA2824, and RetS) transferred the phosphoryl group to HptB (PA3345). The in vivo interaction between HptB and each of the sensors was also confirmed using the bacterial two-hybrid assay. Interestingly, the phosphoryl groups from these sensors all appeared to be transferred via HptB to PA3346, a novel phosphatase consisting of an N-terminal receiver domain and a eukaryotic type Ser/Thr phosphatase domain, and resulted in a significant increase of its phosphatase activity. The subsequent reverse transcription-PCR analysis revealed an operon structure of hptB-PA3346 -PA3347, suggesting a coordinate expression of the three genes to carry out a signal transduction. The possibility was supported by the analysis showing PA3347 is able to be phosphorylated on Ser-56, and this phosphoryl group could be removed by PA3346 protein. Finally, analysis of PA3346 and PA3347 gene knock-out mutants revealed that these genes are associated with bacterial swarming activity and biofilm formation. Together, these results disclose a novel multistep phosphorelay system that is essential for P. aeruginosa to respond to a wide spectrum of environmental signals.Most bacteria possess multiple sets of two-component regulatory systems (2CSs), 2 which are used for the reception of and response to environmental challenges (1). Typical 2CSs are composed of a sensor and a response regulator. The sensor is normally a transmembrane histidine kinase that detects a specific environmental stimulus and auto-phosphorylates a conserved histidine residue within its transmitter domain. The phosphoryl group is subsequently transferred from the histidine residue to an aspartic acid on the cognate regulator, which then activates the expression of genes required for countering the environmental stress. In addition to this basic "His 3 Asp" type of phosphotransfer mechanism, more complex multistep phosphorelay 2CSs also exist, in which the sensor harbors two extra domains as follows: a receiver domain containing a phosphor-accepting Asp and a Hpt domain. The most well characterized examples of the complex type 2CS are the anaerobic regulator ArcAB of Escherichia coli (2) and virulence-associated regulator BvgAS of Bordetella spp. (3). Both are capable of performing a multistep His 3 Asp 3 His 3 Asp phosphorelay.An intermediate group of sensors are known as the hybrid sensors. The hybrid-type sensors, which contain a kinase and a receiver domain but lack an Hpt domain, are believed to require another protein to provide the Hpt domain for their signal transduction ...

Background: This study investigates how histidine phosphotransfer protein-B (HptB) regulates Pseudomonas aeruginosa swarming. Results: HptB regulates the protein phosphatase activity of PA3346, which in turn controls the flagellar gene expression through interaction with PA3347. Conclusion: Our results reveal a partner-switching mechanism regulating the 28 -dependent motility genes. Significance: The interplay between a two-component system and 28 has been established.

Tylophorine-based compounds exert broad spectral, potent inhibition of coronaviruses. NF-κB activation is a common pro-inflammatory response of host cells to viral infection. The aims of this study were to (i) find an effective combination treatment for coronaviral infections through targeting of the virus per se and cellular NF-κB activity; and (ii) to study the underling mechanisms. We found that tylophorine-based compounds target the TGEV viral RNA and effectively inhibit TGEV replication. NF-κB inhibition also leads to anti-TGEV replication. NF-κB activation induced by TGEV infection was found to be associated with two convergent pathways, IKK-2_IκBα/p65 and JAK2 mediated p65 phosphorylation, in swine testicular cells. JAK2 inhibition either by CYT387 (a JAK family inhibitor) or by silencing JAK2-expression revealed a dominant JAK2 mediated p65 phosphorylation pathway for NF-κB activation and resulted in NF-κB inhibition, which overrode the IκBα regulation via the IKK-2. Finally, tylophorine-based compounds work cooperatively with CYT387 to impart comprehensive anti-TGEV activities. The combination treatment, wherein a tylophorine compound targets TGEV and a JAK2 inhibitor blocks the alternative dominant NF-κB activation mediated by JAK2, is more effective and comprehensive than either one alone and constitutes a feasible approach for the treatment of SARS-CoV or MERS-CoV.

A novel in vivo expression technology (IVET) was performed to identify Klebsiella pneumoniae CG43 genes that are specifically expressed during infection of BALB/c mice. The IVET employed a UDP glucose pyrophosphorylase (galU)-deficient mutant of K. pneumoniae which is incapable of utilizing galactose and synthesizing capsular polysaccharide, as demonstrated by its low virulence to BALB/c mice and a white nonmucoid colony morphology on MacConkey-galactose agar. By using a functional galU gene as the reporter, an IVE promoter could render the galU mutant virulent while maintaining the white nonmucoid colony phenotype. A total of 20 distinct sequences were obtained through the in vivo selection. Five of them have been identified previously as virulence-associated genes in other pathogens, while another five with characterized functions are involved in regulation and transportation of nutrient uptake, biosynthesis of isoprenoids, and protein folding. No known functions have been attributed to the other 10 sequences. We have also demonstrated that 2 of the 20 IVE genes turn on under iron deprivation, whereas the expression of another five genes was found to be activated in the presence of paraquat, a superoxide generator.Klebsiella pneumoniae is an important nosocomial pathogen that causes a wide range of infections, including pneumonia, bacteremia, urinary tract infections, and sometimes life-threatening septic shock. As an opportunistic pathogen, it primarily attacks immunocompromised individuals who are hospitalized and/or suffering from severe underlying diseases, such as diabetes mellitus, chronic alcoholism, or pulmonary obstruction (23). Many clinical strains of K. pneumoniae are highly resistant to antibiotics, indicating the relative ineffectiveness of current therapy.During infections, bacterial pathogens must adapt to various changes in order to persist and proliferate in appropriate locations and to circumvent host defenses. It is reasonable to assume that the expression of many K. pneumoniae genes that participate in pathogenesis could be specifically induced within the host. Ideally, these in vivo-expressed (IVE) genes would serve as useful drug targets and vaccine candidates. Several approaches, including in vivo expression technology (IVET) (11, 15), comparative genomics (2), microarray DNA chips (7), signature-tagged mutagenesis (18, 26), differential display-PCR (1), and differential fluorescence detection (31), have allowed the identification of genes that are essential or specifically activated during infections. Nevertheless, none of these approaches has been applied to K. pneumoniae, primarily due to the limited number of mutants and genetic tools available for the bacterium.IVE technology (IVET) is a powerful technique that has been used successfully for several important pathogens, including Salmonella enterica serovar Typhimurium (15, 16), Yersinia enterocolitica (33), Staphylococcus aureus (14), Pseudomonas aeruginosa (32), Escherichia coli (12), and Actinobacillus pleuropneumoniae (9). The ...