Autoimmune causes of encephalitis syndrome in Thailand: prospective study of 103 patients

Background: Data on encephalitis in Thailand have not been completely described. Etiologies remain largely unknown. We prospectively analyzed 103 Thai patients from 27 provinces for the causes of encephalitis using clinical, microbiological and neuroimaging indices; caseswithout a diagnosis were evaluated for autoimmune causes of encephalitis. Methods: Patients with encephalitis and/or myelitis were prospectively studied between October 2010 and August 2012. Cases associated with bacterial, rickettsial and mycobacterial diseases were excluded. Herpes viruses 1-6 and enteroviruses infection was diagnosed using PCR evaluation of CSF; dengue and JE viruses infection, by serology. The serum of test-negative patients was evaluated for the presence of autoantibodies. Results: 103 patients were recruited. Fifty-three patients (52%) had no etiologies identified. Twenty-five patients (24%) were associated with infections. Immune encephalitis was found in 25 (24%); neuropsychiatric lupus erythematosus (4), demyelinating diseases (3), Behcet’s disease (1) and the remaining had antibodies to NMDAR (5), ANNA-2 (6), Yo (2), AMPA (1), GABA (1), VGKC (1) and NMDA coexisting with ANNA-2 (1). Presenting symptoms in the autoimmune group included behavioral changes in 6/25 (versus 12/25 in infectious and 13/53 in unknown group) and as psychosis in 6/25 (versus 0/25 infectious and 2/53 unknown). Seizures were found in 6/25 autoimmune, 4/25 infectious and 19/53 unknown group. Two patients with anti-ANNA-2 and one anti-Yo had temporal lobe involvement by magnetic resonance imaging. Two immune encephalitis patients with antibodies to NMDAR and ANNA-2 had ovarian tumors. Conclusions: Autoantibody-associated encephalitis should be considered in the differential diagnosis and management algorithm regardless of clinical and neuroimaging features

Roles of Agrobacterium tumefaciens RirA in Iron Regulation, Oxidative Stress Response, and Virulence▿

The analysis of genetics and physiological functions of Agrobacterium tumefaciens RirA (rhizobial iron regulator) has shown that it is a transcription regulator and a repressor of iron uptake systems. The rirA mutant strain (NTLrirA) overproduced siderophores and exhibited a highly constitutive expression of genes involved in iron uptake (fhuA, irp6A, and fbpA) compared to that of the wild-type strain (NTL4). The deregulation in the iron control of iron uptake in NTLrirA led to iron overload in the cell, which was supported by the observation that the NTLrirA mutant was more sensitive than wild-type NTL4 to an iron-activated antibiotic, streptonigrin. The NTLrirA mutant was more sensitive than the parental strain to oxidants, including hydrogen peroxide, organic hydroperoxide, and a superoxide generator, menadione. However, the addition of an iron chelator, 2,2′-dipyridyl, reversed the mutant hypersensitivity to H2O2 and organic hydroperoxide, indicating the role of iron in peroxide toxicity. Meanwhile, the reduced level of superoxide dismutase (SodBIII) was partly responsible for the menadione-sensitive phenotype of the NTLrirA mutant. The NTLrirA mutant showed a defect in tumorigenesis on tobacco leaves, which likely resulted from the increased sensitivity of NTLrirA to oxidants and the decreased ability of NTLrirA to induce virulence genes (virB and virE). These data demonstrated that RirA is important for A. tumefaciens during plant-pathogen interactions

Time course of <i>t</i>-BOOH degradation by various <i>PA</i> strains.

<p>The rate of PA <i>t</i>-BOOH degradation, an organic hydroperoxide, was investigated using xylenol orange–iron reaction system as described in the materials and methods section. The exponential phase of PAO1 (white bar), <i>oxyR</i> (light gray bar), <i>oxyR</i>/p<i>oxyR</i> (dotted bar), <i>phnW</i>::Gm/p<i>phnW</i> (black bar) and <i>phnW</i>::Gm (dark gray bar) cells were used in this study in the present of 200 μM of <i>t</i>-BOOH and measured the rate of degradation of <i>t</i>-BOOH in each cell at difference time points. The percentage of <i>t</i>-BOOH remaining was reported after 12 min of incubation. The experiments were independently repeated at least three times and typical results are shown.</p

A model of <i>phnW</i> expression under difference conditions.

<p><b>(A).</b> In wild-type bacteria, <i>phnW</i> is continuously expressed in both untreated bacteria and during exposure to <i>t</i>-BOOH, while oxidized OxyR triggers activation of <i>ahpC</i> expression as previously shown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189066#pone.0189066.ref006" target="_blank">6</a>]. Wild-type bacteria used both of these proteins and Ohr to assist in either the direct or indirect degradation of <i>t</i>-BOOH. This results in efficient <i>t</i>-BOOH degradation to a by-product that is not toxic to the bacteria, thereby preventing damage to proteins, DNA and lipid. (<b>B).</b> Expression of the <i>phnW</i> gene is significant lowered in <i>oxyR</i> mutant bacteria (80%) when compared to wild-type bacteria with no induction of <i>ahpC</i> expression when exposed to <i>t</i>-BOOH. This results in greater susceptibility to <i>t</i>-BOOH of this mutant relative to wild-type bacteria since this strain likely only uses Ohr to help detoxify <i>t</i>-BOOH. (<b>C).</b> Lower expression of <i>phnW</i> was also detected in a PAO1 <i>ahpC</i> mutant compared to wild type bacteria (30%). A lack of this major AHP in response to <i>t</i>-BOOH coupled with lower expression of <i>phnW</i> with only Ohr detoxifying power remaining results in less protection of this mutant from <i>t</i>-BOOH toxicity. (<b>D).</b> An <i>ahpC ohr</i> mutant showed a slightly reduced expression of <i>phnW</i> when bacteria were exposed to <i>t</i>-BOOH, but no difference were observed under control conditions when compared to wild-type expression. Thus, a lack of both of the major <i>t</i>-BOOH detoxification proteins and a lower expression of <i>phnW</i> results in this mutant being the most susceptible to <i>t</i>-BOOH. This may aid in bacterial protection from endogenous free radicals that are continuously generated under aerobic conditions and/or at the earliest time period, when exposed to <i>t</i>-BOOH while the responding gene is not yet expressed. When the bacteria are exposed to <i>t</i>-BOOH, oxidized OxyR governs over-expression of AhpCF to help in its detoxification and together with Ohr, an OxyR independent <i>t</i>-BOOH detoxification protein (<b>Fig 7A</b>). A significantly lower expression level of <i>phnW</i> gene (~80%) was revealed in the <i>oxyR</i> mutant under both reduced and oxidized conditions, indicative of OxyR-mediated regulation of the <i>phnW</i> gene (<b>Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189066#pone.0189066.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189066#pone.0189066.g001" target="_blank">1</a></b>). When the <i>oxyR</i> mutant that had significantly reduced expression of AhpC and PhnW was exposed to <i>t</i>-BOOH, this event triggered an increased susceptibility to this oxidant when compared to wild-type bacteria (<b>Fig 7B</b>). Interestingly, <i>phnW</i> expression levels were also ~30% lower when compared to wild-type levels but are complemented when exposed to <i>t</i>-BOOH in the <i>ahpC</i> but not in the <i>ohr</i> mutant (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189066#pone.0189066.g002" target="_blank">Fig 2</a></b>). This could be yet another mechanism by which bacteria used to protect themselves from <i>t</i>-BOOH toxicity when they lack the major <i>t</i>-BOOH detoxifying protein, AhpC (<b>Fig 7C</b>). Both <i>PA oxyR</i> and <i>PA ahpC</i> mutants still have an Ohr (organic hydroperoxide resistance), one of the major proteins that can contribute to <i>t</i>-BOOH detoxification. A genome search for “peroxidase” and “hydroperoxide” revealed 6 and 5 hits, respectively. This indicates that there are likely multiple redundant mechanisms to dispose of hyperoxides such as <i>t</i>-BOOH. Therefore, we expected that <i>phnW</i> expression should be higher in the <i>ohr ahpC</i> double mutant to protect bacteria cell from <i>t</i>-BOOH. Surprisingly, expression was ~20% lower in this strain after exposure to <i>t</i>-BOOH. The lower expression of PhnW after exposure to <i>t</i>-BOOH in the <i>ohr ahpC</i> double mutant may also contribute to the sensitivity of this double mutant to <i>t</i>-BOOH (<b>Fig 7D</b>). AhpC is OxyR-dependenct but Ohr is not. Therefore, it appears that <i>phnW</i> expression levels depend on the present of OxyR in the cell and also the level of AhpC in <i>ahpC</i> or <i>ahpC ohr</i> mutants. Therefore, it is likely that OxyR directly regulated <i>phnW</i> expression level through binding on an upstream sequence of this gene in certain condition to help protect cell from <i>t</i>-BOOH toxicity (<b>Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189066#pone.0189066.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189066#pone.0189066.g004" target="_blank">4</a></b>). Frustratingly, though the mechanism of PhnW in responding to <i>t</i>-BOOH or regulated by OxyR is still unclear, this study clearly indicates that this protein has the ability to assist in the protection of <i>PA</i> cell from <i>t</i>-BOOH toxicity.</p

Semi-quantitative expression of PA <i>phnW</i>.

<p>Total RNA was isolated from exponential phase <i>PA</i> PAO1 or its isogenic <i>oxyR</i> mutant. Then, 1 μl of cDNA was used to amplify the <i>phnW</i> promoter region with specific primers. The <i>omlA</i> gene was used as an internal constitutive control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189066#pone.0189066.ref014" target="_blank">14</a>] and <i>ahpC</i> was used as a positive gene under OxyR control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189066#pone.0189066.ref006" target="_blank">6</a>].</p

Localization of the OxyR binding domain of the <i>phnW</i> promoter by DNase I footprinting analysis.

<p><b>(</b><u><b>A).</b></u> The 199-bp upstream sequence of <i>phnW</i> containing a putative OxyR domain was used to treat with DNase I in the presence of OxyR at 0, 500 and 1000 nM. The digested DNA was analyzed on a 5% denaturing polyacrylamide gel, followed by autoradiography. The sequence to which OxyR binds on this fragment was identified. (<u><b>B).</b></u> The upstream sequences of <i>phnW</i> that are underlined are the primers used in this study. The bold letters indicate the OxyR binding domain within the <i>phnW</i> upstream sequence while the lower case bold letters indicate the putative OxyR binding site based upon a match to the consensus ATAG-N₇-CTAT-N₇-ATAG-N₇-CTAT sequence used to search for OxyR-dependent gene candidates. The underlined and bold ATG indicates the translational initiation codon of the <i>phnW</i> gene.</p

The OxyR-regulated <i>phnW</i> gene encoding 2-aminoethylphosphonate:pyruvate aminotransferase helps protect <i>Pseudomonas aeruginosa</i> from <i>tert</i>-butyl hydroperoxide

<div><p>The LysR member of bacterial transactivators, OxyR, governs transcription of genes involved in the response to H₂O₂ and organic (alkyl) hydroperoxides (AHP) in the Gram-negative pathogen, <i>Pseudomonas aeruginosa</i>. We have previously shown that organisms lacking OxyR are rapidly killed by <2 or 500 mM H₂O₂ in planktonic and biofilm bacteria, respectively. In this study, we first employed a bioinformatic approach to elucidate the potential regulatory breadth of OxyR by scanning the entire <i>P</i>. <i>aeruginosa</i> PAO1 genome for canonical OxyR promoter recognition sequences (ATAG-N₇-CTAT-N₇-ATAG-N₇-CTAT). Of >100 potential OxyR-controlled genes, 40 were strategically selected that were <u><b><i>not</i></b></u> predicted to be involved in the direct response to oxidative stress (e.g., catalase, peroxidase, etc.) and screened such genes by RT-PCR analysis for potentially positive or negative control by OxyR. Differences were found in 7 of 40 genes when comparing an <i>oxyR</i> mutant vs. PAO1 expression that was confirmed by ß-galactosidase reporter assays. Among these, <i>phnW</i>, encoding 2-aminoethylphosphonate:pyruvate aminotransferase, exhibited reduced expression in the <i>oxyR</i> mutant compared to wild-type bacteria. Electrophoretic mobility shift assays indicated binding of OxyR to the <i>phnW</i> promoter and DNase I footprinting analysis also revealed the sequences to which OxyR bound. Interestingly, a <i>phnW</i> mutant was more susceptible to <i>t</i>-butyl-hydroperoxide (<i>t</i>-BOOH) treatment than wild-type bacteria. Although we were unable to define the direct mechanism underlying this phenomenon, we believe that this may be due to a reduced efficiency for this strain to degrade <i>t</i>-BOOH relative to wild-type organisms because of modulation of AHP gene transcription in the <i>phnW</i> mutant.</p></div

Determination of <i>phnW</i> expression levels in wild-type and mutant strains when exposed to <i>t</i>-BOOH.

<p>All bacteria which contained a <i>phnW</i>-<i>lacZ</i> transcriptional fusion plasmid (pQF-<i>phnW</i>) were grown to exponential phase and exposed to 250 μM <i>t</i>-BOOH. ß-galactosidase activity assays were reported as the mean +/- standard error compared to untreated bacteria. The assays were performed using three independent experiments. The white bars represent untreated bacteria while the gray bars represent <i>t</i>-BOOH treated organisms, respectively.</p