Inter-species lateral gene transfer focused on the Chlamydia plasticity zone identifies loci associated with immediate cytotoxicity and inclusion stability.

Chlamydia muridarum actively grows in murine mucosae and is a representative model of human chlamydial genital tract disease. In contrast, C. trachomatis infections in mice are limited and rarely cause disease. The factors that contribute to these differences in host adaptation and specificity remain elusive. Overall genomic similarity leads to challenges in the understanding of these significant differences in tropism. A region of major genetic divergence termed the plasticity zone (PZ) has been hypothesized to contribute to the host specificity. To evaluate this hypothesis, lateral gene transfer was used to generate multiple hetero-genomic strains that are predominately C. trachomatis but have replaced regions of the PZ with those from C. muridarum. In vitro analysis of these chimeras revealed C. trachomatis-like growth as well as poor mouse infection capabilities. Growth-independent cytotoxicity phenotypes have been ascribed to three large putative cytotoxins (LCT) encoded in the C. muridarum PZ. However, analysis of PZ chimeras supported that gene products other than the LCTs are responsible for cytopathic and cytotoxic phenotypes. Growth analysis of associated chimeras also led to the discovery of an inclusion protein, CTL0402 (CT147), and homolog TC0424, which was critical for the integrity of the inclusion and preventing apoptosis.

Resolution of Radiation-Induced Necrosis in Arteriovenous Malformation with Bevacizumab: A Case Report and Review of Current Literature.

Stereotactic radiosurgery (SRS) is a proven treatment modality for inoperable arteriovenous malformations (AVMs). However, the rate of radiation-induced necrosis (RIN) is as high as 10%. A 6-year-old female patient presented with severe headache, emesis, and syncope, and workup revealed a Spetzler-Martin grade 4 AVM with intraventricular hemorrhage and hydrocephalus. The patient underwent a right frontal ventriculostomy followed by a linear accelerator-based SRS of 16.9 Gy. At 19 years, she developed progressive neurological symptoms. Diagnostic magnetic resonance imaging (MRI) revealed a recurrent parietal AVM nidus. We delivered the linear accelerator-based SRS of 18.5 Gy to the AVM nidus. Within 9 months, she experienced episodic headaches and left-sided weakness and spasticity; symptoms were initially managed with dexamethasone. Follow-up MRI was notable for edema and nondetectable blood flow, consistent with RIN and AVM obliteration. The second course of steroids did not provide the symptom control. Persistent RIN was noted on MRI, and she had stigmata of steroid toxicity (centripetal obesity, depression, and sleep disorder). Two infusions of bevacizumab (5 mg/kg) were administered concurrently with a tapering dose of dexamethasone. The patient noted a near immediate improvement in her headaches, and 2 months following the second bevacizumab infusion, she reported a near-complete resolution of her symptoms and displayed improved ambulation. The development of RIN remains a noteworthy concern post-SRS of AVMs. While steroids aid with initial management of RIN, for persistent and recurrent symptoms, bevacizumab infusions serve as a viable treatment course, with the added benefit of reducing the likelihood of adverse effects resulting from prolonged steroid therapy.

Development of Transposon Mutagenesis for Chlamydia muridarum.

Functional genetic analysis of Chlamydia has been a challenge due to the historical genetic intractability of Chlamydia, although recent advances in chlamydial genetic manipulation have begun to remove these barriers. Here, we report the development of the Himar C9 transposon system for Chlamydia muridarum, a mouse-adapted Chlamydia species that is widely used in Chlamydia infection models. We demonstrate the generation and characterization of an initial library of 33 chloramphenicol (Cam)-resistant, green fluorescent protein (GFP)-expressing C. muridarum transposon mutants. The majority of the mutants contained single transposon insertions spread throughout the C. muridarum chromosome. In all, the library contained 31 transposon insertions in coding open reading frames (ORFs) and 7 insertions in intergenic regions. Whole-genome sequencing analysis of 17 mutant clones confirmed the chromosomal locations of the insertions. Four mutants with transposon insertions in glgB, pmpI, pmpA, and pmpD were investigated further for in vitro and in vivo phenotypes, including growth, inclusion morphology, and attachment to host cells. The glgB mutant was shown to be incapable of complete glycogen biosynthesis and accumulation in the lumen of mutant inclusions. Of the 3 pmp mutants, pmpI was shown to have the most pronounced growth attenuation defect. This initial library demonstrates the utility and efficacy of stable, isogenic transposon mutants for C. muridarum The generation of a complete library of C. muridarum mutants will ultimately enable comprehensive identification of the functional genetic requirements for Chlamydia infection in vivoIMPORTANCE Historical issues with genetic manipulation of Chlamydia have prevented rigorous functional genetic characterization of the ∼1,000 genes in chlamydial genomes. Here, we report the development of a transposon mutagenesis system for C. muridarum, a mouse-adapted Chlamydia species that is widely used for in vivo investigations of chlamydial pathogenesis. This advance builds on the pioneering development of this system for C. trachomatis We demonstrate the generation of an initial library of 33 mutants containing stable single or double transposon insertions. Using these mutant clones, we characterized in vitro phenotypes associated with genetic disruptions in glycogen biosynthesis and three polymorphic outer membrane proteins.