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Methionine aminopeptidase (MetAP) is a class of ubiquitous enzymes essential for the survival of numerous bacterial species. These enzymes are responsible for the cleavage of N-terminal formyl-methionine initiators from nascent proteins to initiate post-translational modifications that are often essential to proper protein function. Thus, inhibition of MetAP activity has been implicated as a novel antibacterial target. We tested this idea in the present study by targeting the MetAP enzyme in the obligate intracellular pathogen Rickettsia prowazekii. We first identified potent RpMetAP inhibitory species by employing an in vitro enzymatic activity assay. The molecular docking program AutoDock was then utilized to compare published crystal structures of inhibited MetAP species to docked poses of RpMetAP. Based on these in silico and in vitro screens, a subset of 17 compounds was tested for inhibition of R. prowazekii growth in a pulmonary vascular endothelial cell (EC) culture infection model system. All compounds were tested over concentration ranges that were determined to be non-toxic to the ECs and 8 of the 17 compounds displayed substantial inhibition of R. prowazekii growth. These data highlight the therapeutic potential for inhibiting RpMetAP as a novel antimicrobial strategy and set the stage for future studies in pre-clinical animal models of infection.

Background: Bacterial pneumonia and sepsis are both common causes of end-organ dysfunction, especially in immunocompromised and critically ill patients. Pre-clinical data demonstrate that bacterial pneumonia and sepsis elicit the production of cytotoxic tau and amyloids from pulmonary endothelial cells, which cause lung and brain injury in naïve animal subjects, independent of the primary infection. The contribution of infection-elicited cytotoxic tau and amyloids to end-organ dysfunction has not been examined in the clinical setting. We hypothesized that cytotoxic tau and amyloids are present in the bronchoalveolar lavage fluid of critically ill patients with bacterial pneumonia and that these tau/amyloids are associated with end-organ dysfunction. Methods: Bacterial culture-positive and culture-negative mechanically ventilated patients were recruited into a prospective, exploratory observational study. Levels of tau and Aβ42 in, and cytotoxicity of, the bronchoalveolar lavage fluid were measured. Cytotoxic tau and amyloid concentrations were examined in comparison with patient clinical characteristics, including measures of end-organ dysfunction. Results: Tau and Aβ42 were increased in culture-positive patients (n = 49) compared to culture-negative patients (n = 50), independent of the causative bacterial organism. The mean age of patients was 52.1 ± 16.72 years old in the culture-positive group and 52.78 ± 18.18 years old in the culture-negative group. Males comprised 65.3% of the culture-positive group and 56% of the culture-negative group. Caucasian culture-positive patients had increased tau, boiled tau, and Aβ42 compared to both Caucasian and minority culture-negative patients. The increase in cytotoxins was most evident in males of all ages, and their presence was associated with end-organ dysfunction. Conclusions: Bacterial infection promotes the generation of cytotoxic tau and Aβ42 within the lung, and these cytotoxins contribute to end-organ dysfunction among critically ill patients. This work illuminates an unappreciated mechanism of injury in critical illness. [ABSTRACT FROM AUTHOR]

Rickettsia prowazekii is an obligate intracellular pathogen that possesses a small genome and a highly refined repertoire of biochemical pathways compared to those of free-living bacteria. Here we describe a novel biochemical pathway that relies on rickettsial transport of host cytosolic dihydroxyacetone phosphate (DHAP) and its subsequent conversion to sn-glycerol-3-phosphate (G3P) for synthesis of phospholipids. This rickettsial pathway compensates for the evolutionary loss of rickettsial glycolysis/gluconeogenesis, the typical endogenous source of G3P. One of the components of this pathway is R. prowazekii open reading frame RP442, which is annotated GpsA, a G3P dehydrogenase (G3PDH). Purified recombinant rickettsial GpsA was shown to specifically catalyze the conversion of DHAP to G3P in vitro. The products of the GpsA assay were monitored spectrophotometrically, and the identity of the reaction product was verified by paper chromatography. In addition, heterologous expression of the R. prowazekii gpsA gene functioned to complement an Escherichia coli gpsA mutant. Furthermore, gpsA mRNA was detected in R. prowazekii purified from hen egg yolk sacs, and G3PDH activity was assayable in R. prowazekii lysed-cell extracts. Together, these data strongly suggested that R. prowazekii encodes and synthesizes a functional GpsA enzyme, yet R. prowazekii is unable to synthesize DHAP as a substrate for the GpsA enzymatic reaction. On the basis of the fact that intracellular organisms often avail themselves of resources in the host cell cytosol via the activity of novel carrier-mediated transport systems, we reasoned that R. prowazekii transports DHAP to supply substrate for GpsA. In support of this hypothesis, we show that purified R. prowazekii transported and incorporated DHAP into phospholipids, thus implicating a role for GpsA in vivo as part of a novel rickettsial G3P acquisition pathway for phospholipid biosynthesis. doi: 10.1128/JB.00443-10

The obligate intracytoplasmic pathogen Rickettsia prowazekii relies on the transport of many essential compounds from the cytoplasm of the eukaryotic host cell in lieu of de novo synthesis, an evolutionary outcome undoubtedly linked to obligatory growth in this metabolite-replete niche. The paradigm for the study of rickettsial transport systems is the ATP/ADP translocase Tlc1, which exchanges bacterial ADP for host cell ATP as a source of energy, rather than as a source of adenylate. Interestingly, the R. prowazekii genome encodes four open reading frames that are highly homologous to the well-characterized ATP/PADP translocase Tlc1. Therefore, by annotation, the R. prowazekii genome encodes a total of five ATP/ADP translocases: Tlc1, Tlc2, Tlc3, Tlc4, and Tlc5. We have confirmed by quantitative reverse transcriptase PCR that mRNAs corresponding to all five tic homologues are expressed in R. prowazekii growing in L-929 cells and have shown their heterologous protein expression in Escherichia coli, suggesting that none of the tic genes are pseudogenes in the process of evolutionary meltdown. However, we demonstrate by heterologous expression in E. coli that only Tlc1 functions as an ATP/ADP transporter. A survey of nucleotides and nucleosides has determined that Tlc4 transports CTP, UTP, and GDP. Intriguingly, although GTP was not transported by Tlc4, it was an inhibitor of CTP and UTP uptake and demonstrated a [K.sub.i] similar to that of GDP. In addition, we demonstrate that Tlc5 transports GTP and GDP. We postulate that Tlc4 and Tlc5 serve the primary function of maintaining intracellular pools of nucleotides for rickettsial nucleic acid biosynthesis and do not provide the cell with nucleoside triphosphates as an energy source, as is the case for Tlc1. Although heterologous expression of Tlc2 and Tlc3 was observed in E. coli, we were unable to identify substrates for these proteins.

The accessibility of the Rickettsia prowazekii ATP/ADP translocase transmembrane domains (TMs) IV-VII and IX-XII to the putative, water-filled ATP translocation pathways is determined. The preexposure of many methanethiosulfonate (MTS)-sensitive mutants in TMs V, VII, X and XI to ATP concentrations 10 times the K(sub m) results in protection from MTS-mediated inhibition, thereby confirming exposure of these TMs to the aqueous ATP translocation pathway.

The contribution of transmembrane regions I, II, and III of the Rickettsia prowazekii ATP/ADP translocase to the structure of the putative water-filled ATP translocation channel is determined from the accessibility of hydrophilic, thiol-reactive, methanethiosulfonate reagents to a library of 68 independent cycteine-substitution mutants heterologously expressed in Escherichia coli. Based on the lack of accessibility of charged MTS reagents to the thiol groups in mutants of TM III, it appears that TM II is not exposed to the ATP translocation channel.

The two obligate intracellular alphaproteobacteria Rickettsia prowazekii and Caedibacter caryophilus, a human pathogen and a paramecium endosymbiont, respectively, possess transport systems to facilitate ATP uptake from the host cell cytosol. These transport proteins, which have 65% identity at the amino acid level, were heterologously expressed in Escherichia coli, and their properties were compared. The results presented here demonstrate that the caedibacter transporter had a broader substrate than the more selective rickettsial transporter. ATP analogs with modified sugar moieties, dATP and ddATP, inhibited the transport of ATP by the caedibacter transporter but not by the rickettsial transporter. Both transporters were specific for di- and trinucleotides with an adenine base in that adenosine tetraphosphate, AMP, UTP, CTP, and GTP were not competitive inhibitors. Furthermore, the antiporter nature of both transport systems was shown by the dependence of the efflux of [[[alpha]-.sup.32]P]ATP on the influx of substrate (ATP but not dATP for rickettsiae, ATP or dATP for caedibacter).

Research was conducted to identify the conserved, RpoS-dependent stationary-phase genes of Escherichia coli using a mutant identification scheme where an rpoS null allele is introduced into strains containing random promoter-lacZ fusions. A genetic screen was used to isolate over 100 independent promoter-lacZ operon fusion mutations. DNA sequence analysis determined the identities of the 10 most highly Rpo-S-dependent fusions identified in the Escherichia coli.

The evaluation of the contribution of transmembrane region VIII of the Rickettsia prowazekii ATP/ADP translocase to the structure of the water filled channel through which ATP is transported is discussed. The results indicate that TM VIII makes a major contribution to the structure of the aqueous translocation pathway, that the accessibility to impermeant thiol reagents is influenced by substrate, and that there is great variation in accessibility to MTS reagents along the length of TM VIII.