Phylogenetic relationship of <i>Enterocytozoon bieneusi</i> groups, the relationship between <i>E</i>. <i>bieneusi</i> genotypes identified in this study and other known genotypes deposited in the GenBank was inferred by a neighbor-joining analysis of ITS sequences based on genetic distance by the Kimura-2-parameter model.

<p>The numbers on the branches represent percent bootstrapping values from 1,000 replicates, with more than 50% shown in tree. Each sequence is identified by its accession number, genotype designation, and host origin. The group terminology for the clusters is based on the work of Zhao et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163605#pone.0163605.ref028" target="_blank">28</a>]. Genotypes with <i>black circles</i> and <i>open circles</i> are novel and known genotypes identified in this study, respectively.</p

Gene locus, primer sequences, annealing temperatures and fragment length for the identification of <i>E</i>. <i>bieuensi</i> used in this study.

<p>Gene locus, primer sequences, annealing temperatures and fragment length for the identification of <i>E</i>. <i>bieuensi</i> used in this study.</p

First Report of the Human-Pathogenic <i>Enterocytozoon bieneusi</i> from Red-Bellied Tree Squirrels (<i>Callosciurus erythraeus</i>) in Sichuan, China

<div><p><i>Enterocytozoon bieneusi</i> is a common opportunistic pathogen causing diarrhea and enteric disease in a variety of animal hosts. Although it has been reported in many animals, there is no published information available on the occurrence of <i>E</i>. <i>bieneusi</i> in red-bellied tree squirrels. To understand the occurrence, genetic diversity, and zoonotic potential of <i>E</i>. <i>bieneusi</i> in red-bellied tree squirrels, 144 fecal specimens from Sichuan province, China, were examined by PCR amplification and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of <i>E</i>. <i>bieneusi</i>. The overall infection rate of <i>E</i>. <i>bieneusi</i> 16.7% (24/144) was observed in red-bellied tree squirrels. Altogether five genotypes of <i>E</i>. <i>bieneusi</i> were identified: three known genotypes D (n = 18), EbpC (n = 3), SC02 (n = 1) and two novel genotypes CE01, CE02 (one each). Multilocus sequence typing (MLST) analysis employing three microsatellite (MS1, MS3, MS7) and one minisatellite (MS4) revealed 16, 14, 7 and 14 positive specimens were successfully sequenced, and identified eight, three, three and two genotypes at four loci, respectively. In phylogenetic analysis, the three known genotypes D, EbpC, and SC02 were clustered into group 1 with zoonotic potential, and the two novel genotypes CE01 and CE02 were clustered into group 6. The present study firstly reported the occurrence of <i>E</i>. <i>bieneusi</i> in red-bellied tree squirrels in China, and the <i>E</i>. <i>bieneusi</i> genotypes D and EbpC were found in humans previously. These results indicate that red-bellied tree squirrels may play a potential role in the transmission of <i>E</i>. <i>bieneusi</i> to humans.</p></div

Multilocus characterization of <i>E</i>. <i>bieneusi</i> isolates from red-bellied tree squirrels in Sichuan, southwestern China.

<p>Multilocus characterization of <i>E</i>. <i>bieneusi</i> isolates from red-bellied tree squirrels in Sichuan, southwestern China.</p

Distribution of <i>E</i>. <i>bieneusi</i> genotypes in red-bellied tree squirrels from different countries.

<p>Distribution of <i>E</i>. <i>bieneusi</i> genotypes in red-bellied tree squirrels from different countries.</p

Occurrence and genotypes of <i>E</i>. <i>bieneusi</i> in red-bellied tree squirrels from different cities and sources of southwest China.

<p>Occurrence and genotypes of <i>E</i>. <i>bieneusi</i> in red-bellied tree squirrels from different cities and sources of southwest China.</p

Occurrence and genotypes of <i>E</i>. <i>bieneusi</i> in red-bellied tree squirrels by age and gender.

<p>Occurrence and genotypes of <i>E</i>. <i>bieneusi</i> in red-bellied tree squirrels by age and gender.</p

Impact of Hfq on Global Gene Expression and Intracellular Survival in <i>Brucella melitensis</i>

<div><p><i>Brucella melitensis</i> is a facultative intracellular bacterium that replicates within macrophages. The ability of brucellae to survive and multiply in the hostile environment of host macrophages is essential to its virulence. The RNA-binding protein Hfq is a global regulator that is involved in stress resistance and pathogenicity. Here we demonstrate that Hfq is essential for stress adaptation and intracellular survival in <i>B. melitensis</i>. A <i>B. melitensis hfq</i> deletion mutant exhibits reduced survival under environmental stresses and is attenuated in cultured macrophages and mice. Microarray-based transcriptome analyses revealed that 359 genes involved in numerous cellular processes were dysregulated in the <i>hfq</i> mutant. From these same samples the proteins were also prepared for proteomic analysis to directly identify Hfq-regulated proteins. Fifty-five proteins with significantly affected expression were identified in the <i>hfq</i> mutant. Our results demonstrate that Hfq regulates many genes and/or proteins involved in metabolism, virulence, and stress responses, including those potentially involved in the adaptation of <i>Brucella</i> to the oxidative, acid, heat stress, and antibacterial peptides encountered within the host. The dysregulation of such genes and/or proteins could contribute to the attenuated <i>hfq</i> mutant phenotype. These findings highlight the involvement of Hfq as a key regulator of <i>Brucella</i> gene expression and facilitate our understanding of the role of Hfq in environmental stress adaptation and intracellular survival of <i>B. melitensis</i>.</p></div

Differentially expressed transcripts (upper graphs) and proteins (lower graphs) in the <i>B. melitensis</i> Δhfq mutant strain.

<p>Histograms show the number of differentially expressed genes and their distributions in the <i>B. melitensis</i> chromosome. The functional categories according to the <i>B. melitensis</i> 16 M genome sequence annotation and the KEGG database is shown to the right in circle charts. The number of genes belonging to each category are shown in brackets.</p

Hfq upregulated the expression of Omp25/Omp31 and the Sigma factors.

<p>A. Fold changes in the transcript abundances of <i>omp25</i>, <i>omp25b</i>, <i>omp25c</i>, and <i>omp31</i> genes were detected by microarray and qRT-PCR in 16 MΔhfq, relative to 16 M. B, C. Transcript abundances of <i>rpoH1</i> (B) and <i>rpoE1</i> (C) were detected in the 16 M, 16 MΔhfq, and 16 MΔhfq-C during early logarithmic (EL), mid-logarithmic (ML), and stationary phases (SP). Significant differences between the transcription abundances of <i>rpoH1</i> and <i>rpoE1</i> in the mutant and parent strain are indicated as follows: *, P<0.001.</p