The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.

cWe previously associated a missense mutation of the tc0668 gene of serial in vitro-passaged Chlamydia muridarum, a murine model of human urogenital C. trachomatis, with severely attenuated disease development in the upper genital tract of female mice. Since these mutants also contained a TC0237 Q117E missense mutation that enhances their in vitro infectivity, an effort was made here to isolate and characterize a tc0668 single mutant to determine its individual contribution to urogenital pathogenicity. Detailed genetic analysis of C. muridarum passages revealed a truncated variant with a G216* nonsense mutation of the 408-amino-acid TC0668 protein that does not produce a detectable product. Intracellular growth and infectivity of C. muridarum in vitro remain unaffected in the absence of TC0668. Intravaginal inoculation of the TC0668 null mutant into C3H/HeJ mice results in a typical course of lower genital tract infection but, unlike a pathogenic isogenic control, is unable to elicit significant chronic inflammation of the oviduct and fails to induce hydrosalpinx. Thus, TC0668 is demonstrated as an important chromosome-encoded urogenital pathogenicity factor of C. muridarum and the first with these characteristics to be discovered for a Chlamydia pathogen. Chlamydia muridarum is a Gram-negative obligate intracellular pathogen that was isolated from a steady-state respiratory infection of laboratory mice in the early 1940s (1, 2). Like other chlamydial organisms, C. muridarum has a biphasic life cycle that alternates between infectious elementary body (EB) and replicating reticulate body (RB) morphologies. The genome of C. muridarum is reductively evolved, containing a 1.07-Mb circular chromosome and single 7.5-kb extrachromosomal plasmid (3). In that C. muridarum and other chlamydial pathogens have fewer than 1,000 genes and ϳ900 encoded proteins, roughly half the number encoded by environmental chlamydial organisms that parasitize simple single-celled eukaryotes (4), it is not known which of the many cryptic genetic factors allow them to thrive within and harm complex vertebrates.In the laboratory, C. muridarum is used as a model of urogenital disease resulting from sexually transmitted Chlamydia trachomatis in women. The basic biology and genomes of these two pathogens are highly conserved. The urogenital biovar of C. trachomatis is responsible for the most reported cases of bacterial infection in the United States (5) and is a pervasive global health problem (6). In women, ascending infection from the lower to upper genital tracts, separated by the cervical barrier, can lead to loss of the ciliated epithelium and irreversible fibrotic remodeling of the fallopian tubes after primary infection is resolved (7). If left untreated, often because of asymptomatic infection (8), afflicted women can experience severe chronic sequelae, such as tubal blockage, hydrosalpinx, spontaneous abortion, ectopic pregnancy, and tubal factor infertility (9, 10). Genital inoculation of female mice with C. muridarum results in analo...

bAlthough modern Chlamydia muridarum has been passaged for decades, there are no reports on the consequences of serial passage with strong selection pressure on its fitness. In order to explore the potential for Pasteurian selection to induce genomic and phenotypic perturbations to C. muridarum, a starter population was passaged in cultured cells for 28 generations without standard infection assistance. The resultant population, designated CMG28, displays markedly reduced in vitro dependence on centrifugation for infection and low incidence and severity of upper genital tract pathology following intravaginal inoculation into mice compared to the parental C. muridarum population, CMG0. Deep sequencing of CMG0 and CMG28 revealed novel protein variants in the hypothetical genes TC0237 (Q117E) and TC0668 (G322R). In vitro attachment assays of isogenic plaque clone pairs with mutations in either TC0237 and TC0668 or only TC0237 reveal that TC0237(Q117E) is solely responsible for enhanced adherence to host cells. Paradoxically, double mutants, but not TC0237(Q117E) single mutants, display severely attenuated in vivo pathogenicity. These findings implicate TC0237 and TC0668 as novel genetic factors involved in chlamydial attachment and pathogenicity, respectively, and show that serial passage under selection pressure remains an effective tool for studying Chlamydia pathogenicity. Infection with Chlamydia trachomatis in the lower genital tract (LGT) of women can lead to upper genital tract (UGT) inflammatory pathologies, such as hydrosalpinx, resulting in complications including ectopic pregnancy and infertility (1, 2). Hydrosalpinx, which is detectable by laparoscopic examination, has been used as a surrogate marker for tubal factor infertility in women (3, 4). However, the mechanisms by which C. trachomatis induces hydrosalpinges remain unknown. The murine pathogen Chlamydia muridarum, although not known to cause human diseases, has been extensively used for studying the mechanisms of C. trachomatis pathogenesis and immunity (5-8). This is due primarily to the ease of intravaginal infection of mice with C. muridarum organisms and their ability to induce hydrosalpinx in the oviduct, leading to mouse infertility (5, 9).Both C. trachomatis and C. muridarum share a highly conserved biphasic growth cycle, which begins with the attachment of an infectious elementary body (EB) to a host cell. Multiple putative chlamydial factors, such as the major outer membrane protein (MOMP) (10-13), outer membrane complex (OMC) protein B (OmcB) (14-16), and the polymorphic membrane proteins (Pmps) (17-19), and host-derived factors, such as heparin sulfate (16,20), epidermal growth factor receptor (EGFR) (21), estrogen receptor (22), and insulin-like growth factor 2 receptor (23), have been proposed to mediate chlamydial interactions with host cells. However, the precise structural basis of the interactions between an EB and a host cell during chlamydial infection in animals and humans remains ill defined. Following attachment to epithelia...

We have previously shown that the plasmid-encoded Pgp3 is a major virulence factor for C. muridarum induction of hydrosalpinx. We now report that Pgp5 also plays a significant role in the development of hydrosalpinx following C. muridarum induction. Pgp5 deficiency was introduced via either in-frame deletion (CM-Δpgp5) or premature stop codon installation (CM-pgp5S). Mice infected with either CM-Δpgp5 or CM-pgp5S developed hydrosalpinges at significantly reduced levels with an incidence rate of <40% and a mean severity score of 2 or less. In contrast, 80% or more mice developed hydrosalpinx with a severity score of >3 when mice were infected with Pgp5-sufficient C. muridarum (plasmid-competent wild type or plasmid-free C. muridarum transformed with a full plasmid or depleted of pgp7 gene). The attenuated pathogenicity of the Pgp5-deficient C. muridarum correlated with a significantly reduced level of ascending infection in the oviduct tissue despite the similar overall shedding courses between mice infected with Pgp5-deficeint versus sufficient C. muridarum. Furthermore, in the oviducts of mice infected with Pgp5-deficient C. muridarum, significantly lower levels of inflammatory cell infiltration and cytokine production were detected. Thus, Pgp5 is a significant plasmid-encoded virulence factor for C. muridarum pathogenicity in the upper genital tract.

CPAF is a conserved and secreted protease from obligate intracellular bacteria of the order Chlamydiae. Recently, it was demonstrated that most of its host targets are an artifact of inaccurate methods. This review aims to summarize key features of CPAF and propose new approaches for evaluating its role in chlamydial pathogenesis.

bHydrosalpinx is a pathological hallmark of tubal infertility associated with chlamydial infection. However, the mechanisms of hydrosalpinx remain unknown. Here, we report that complement factor 5 (C5) contributes significantly to chlamydial induction of hydrosalpinx. Mice lacking C5 (C5 ؊/؊ ) failed to develop any hydrosalpinx, while ϳ42% of the corresponding wild-type mice (C5 ؉/؉ ) did so following intravaginal infection with Chlamydia muridarum. Surprisingly, deficiency in C3 (C3 ؊/؊ ), an upstream component of the complement system, did not affect mouse susceptibility to chlamydial induction of hydrosalpinx. Interestingly, C5 activation was induced by chlamydial infection in oviducts of C3 ؊/؊ mice, explaining why the C3 ؊/؊ mice remained susceptible to chlamydial induction of hydrosalpinx. Similar levels of live chlamydial organisms were recovered from oviduct tissues of both C5 ؊/؊ and C5 ؉/؉ mice, suggesting that C5 deficiency did not affect C. muridarum ascending infection. Furthermore, C5؊/؊ mice were still more resistant to hydrosalpinx induction than C5 ؉/؉ mice, even when live C. muridarum organisms were directly delivered into the upper genital tract, both confirming the role of C5 in promoting hydrosalpinx and indicating that the C5-facilitated hydrosalpinx was not due to enhancement of ascending infection. The C5 ؊/؊ mice displayed significantly reduced lumenal inflammatory infiltration and cytokine production in oviduct tissue, suggesting that C5 may contribute to chlamydial induction of hydrosalpinx by enhancing inflammatory responses.

BACKGROUND Next-generation sequencing (NGS) is revolutionizing a variety of molecular biology fields including bioforensics, biosurveillance, and infectious disease diagnostics. For pathogen detection, the ability to sequence all nucleic acids in a sample allows near limitless multiplexability, free from a priori knowledge regarding an etiologic agent as is typically required for targeted molecular assays such as real-time PCR. Furthermore, sequencing capabilities can generate in depth genomic information, allowing detailed molecular epidemiological studies and bioforensics analysis, which is critical for source agent identification in a biothreat outbreak. However, lack of analytical specificity, inherent to NGS, presents challenges for regulated applications such as clinical diagnostics and molecular attribution. CONTENT Here, we discuss NGS applications in the context of preparedness and biothreat readiness. Specifically, we investigate current and future applications of NGS technologies to affect the fields of biosurveillance, bioforensics, and clinical diagnostics with specific focus on biodefense. SUMMARY Overall, there are many advantages to the implementation of NGS for preparedness and readiness against biowarfare agents, from forensics to diagnostics. However, appropriate caveats must be associated with any technology. This includes NGS. While NGS is not the panacea replacing all molecular techniques, it will greatly enhance the ability to detect, characterize, and diagnose biowarfare agents, thus providing an excellent addition to the biodefense toolbox of biosurveillance, bioforensics, and biothreat diagnosis.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a geographically widespread RNA virus with a high degree of genomic diversity that complicates sequence-based diagnostics. Here, we sequenced eight CCHFV strains for improved assay design and deposition into FDA-ARGOS, the FDA’s pathogen database for development and verification of next generation sequencing assays.