live-cell-forward-genetic-approach-to-identify-isolate-developmental

Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in <em>Chlamydia trachomatis</em>

The intracellular bacterial pathogen Chlamydia trachomatis undergoes a developmental cycle consisting of two morphologically discrete developmental forms. ...

University of Idaho

The role of the chlamydial effector CPAF in the induction of genomic instability.

Our laboratory previously reported interesting rods 3-10 μm long and rings 2-5 μm diameter (RR) in the cytoplasm of mammalian cells. Experimental evidence show that both inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) and cytidine triphosphate synthetase (CTPS) are components of RR structures. Several cell types, including mouse embryonic stem cells, and cell lines, such as mouse 3 T3 and rat NRK, naturally present RR structures, while other cells can present RR when treated with compounds interfering with GTP/CTP biosynthetic pathways. In this study, we aimed to investigate the dynamic behavior of these RR in live cells.

Microinjection of specific anti-IMPDH2 antibodies induces disassembly of cytoplasmic rods/rings that are primarily stationary and stable structures.

The non-coding small RNA, IhtA expressed by the obligate intracellular human pathogen Chlamydia trachomatis modulates the translation of HctA, a key protein involved in replicative to infectious cell type differentiation. Using a combination of bioinformatics and mutagenesis we sought to identify the base pairing requirement for functional repression of HctA protein expression, with an eye to applying our findings towards the identification of additional targets. IhtA is predicted to fold into a three stem:loop structure. We found that loop 1 occludes the initiation codon of hctA, while loop 2 and 3 are not required for function. This 7 nucleotide region forms G/C rich interactions surrounding the AUG of hctA. Two additional genes in the chlamydial genome, CTL0322 and CTL0097, contained some elements of the hctA:IhtA recognition sequence. The mRNA of both CTL0322and CTL0097 interacted with IhtA in vitro as measured by biolayer interferometry. However, using a CheZ reporter expression system, IhtA only inhibited the translation of CTL0322. The proposed IhtA recognition site in the CTL0322 message contains significant G/C base pairing on either side of the initiation codon while CTL0097 only contains G/C base pairing 3' to the AUG initiation codon. These data suggest that as the functional interacting region is only 6-7nt in length that full translation repression is dependent on the degree of G/C base pairing. Additionally our results indicate that IhtA may regulate multiple mRNAs involved in the chlamydial infectious cycle. 

Identification of the base-pairing requirements for repression of hctA translation by the small RNA IhtA leads to the discovery of a new mRNA target in Chlamydia trachomatis.

Bacteria of the genus  include the significant human pathogens  and  All chlamydiae are obligate intracellular parasites that depend on infection of a host cell and transition through a biphasic developmental cycle. Following host cell invasion by the infectious elementary body (EB), the pathogen transitions to the replicative but noninfectious reticulate body (RB). Differentiation of the RB back to the EB is essential to generate infectious progeny. While the EB form has historically been regarded as metabolically inert, maintenance of infectivity during incubation with specific nutrients has revealed active maintenance of the infectious phenotype. Using transcriptome sequencing, we show that the transcriptome of extracellular EBs incubated under metabolically stimulating conditions does not cluster with germinating EBs but rather with the transcriptome of EBs isolated directly from infected cells. In addition, the transcriptional profile of the extracellular metabolizing EBs more closely resembled that of EB production than germination. Maintenance of infectivity of extracellular EBs was achieved by metabolizing chemically diverse compounds, including glucose 6-phosphate, ATP, and amino acids, all of which can be found in extracellular environments, including mucosal secretions. We further show that the EB cell type actively maintains infectivity in the inclusion after terminal differentiation. Overall, these findings contribute to the emerging understanding that the EB cell form is actively maintained through metabolic processes after terminal differentiation to facilitate prolonged infectivity within the inclusion and under host cell free conditions, for example, following deposition at mucosal surfaces. Chlamydiae are obligate intracellular Gram-negative bacteria that are responsible for a wide range of diseases in both animal and human hosts. According to the Centers for Disease Control and Prevention,  is the most frequently reported sexually transmitted infection in the United States, costing the American health care system nearly $2.4 billion annually. Every year, there are over 4 million new cases of  infections in the United States and an estimated 100 million cases worldwide. To cause disease,  must successfully complete its complex biphasic developmental cycle, alternating between an infectious cell form (EB) specialized for initiating entry into target cells and a replicative form (RB) specialized for creating and maintaining the intracellular replication niche. The EB cell form has historically been considered metabolically quiescent, a passive entity simply waiting for contact with a host cell to initiate the next round of infection. Recent studies and data presented here demonstrate that the EB maintains its infectious phenotype by actively metabolizing a variety of nutrients. Therefore, the EB appears to have an active role in chlamydial biology, possibly within multiple environments, such as mucosal surfaces, fomites, and inside the host cell after formation.

Article	Year
The role of the chlamydial effector CPAF in the induction of genomic instability. Pathogens and disease\| PubMed ID: 25082267	2014
Microinjection of specific anti-IMPDH2 antibodies induces disassembly of cytoplasmic rods/rings that are primarily stationary and stable structures. Cell & bioscience\| PubMed ID: 25601894	2015
Identification of the base-pairing requirements for repression of hctA translation by the small RNA IhtA leads to the discovery of a new mRNA target in Chlamydia trachomatis. PloS one\| PubMed ID: 25756658	2015
Impact of Active Metabolism on Chlamydia trachomatis Elementary Body Transcript Profile and Infectivity. Journal of bacteriology\| PubMed ID: 29735758	2018

Scott S. Grieshaber

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