Point-counterpoint: Should we be performing metagenomic next-generation sequencing for infectious disease diagnosis in the clinical laboratory?

With established applications of next-generation sequencing in inherited diseases and oncology, clinical laboratories are evaluating the use of metagenomics for identification of infectious agents directly from patient samples, to aid in the diagnosis of infections. Metagenomic next-generation sequencing for infectious diseases promises an unbiased approach to detection of microbes that does not depend on growth in culture or the targeting of specific pathogens. However, the issues of contamination, interpretation of results, selection of databases used for analysis, and prediction of antimicrobial susceptibilities from sequencing data remain challenges. In this Point-Counterpoint, Steve Miller and Charles Chiu discuss the pros of using direct metagenomic sequencing, while Kyle Rodino and Melissa Miller argue for the use of caution

Orientia tsutsugamushi ankyrin repeat-containing protein family members are Type 1 secretion system substrates that traffic to the host cell endoplasmic reticulum

Scrub typhus is an understudied, potentially fatal infection that threatens one billion persons in the Asia-Pacific region. How the causative obligate intracellular bacterium, Orientia tsutsugamushi, facilitates its intracellular survival and pathogenesis is poorly understood. Many intracellular bacterial pathogens utilize the Type 1 (T1SS) or Type 4 secretion system (T4SS) to translocate ankyrin repeat-containing proteins (Anks) that traffic to distinct subcellular locations and modulate host cell processes. The O. tsutsugamushi genome encodes one of the largest known bacterial Ank repertoires plus T1SS and T4SS components. Whether these potential virulence factors are expressed during infection, how the Anks are potentially secreted, and to where they localize in the host cell are not known. We determined that O. tsutsugamushi transcriptionally expresses 20 unique ank genes as well as genes for both T1SS and T4SS during infection of mammalian host cells. Examination of the Anks’ C-termini revealed that the majority of them resemble T1SS substrates. Escherichia coli expressing a functional T1SS was able to secrete chimeric hemolysin proteins bearing the C-termini of 19 of 20 O. tsutsugamushi Anks in an HlyBD-dependent manner. Thus, O. tsutsugamushi Anks C-termini are T1SS-compatible. Conversely, Coxiella burnetii could not secrete heterologously expressed Anks in a T4SS-dependent manner. Analysis of the subcellular distribution patterns of 20 ectopically expressed Anks revealed that, while 6 remained cytosolic or trafficked to the nucleus, 14 localized to, and in some cases, altered the morphology of the endoplasmic reticulum. This study identifies O. tsutsugamushi Anks as T1SS substrates and indicates that many display a tropism for the host cell secretory pathway

Collaboration between clinical and academic laboratories for sequencing SARS-CoV-2 genomes

Genomic sequencing of SARS-CoV-2 continues to provide valuable insight into the ever-changing variant makeup of the COVID-19 pandemic. More than three million SARS-COV-2 genomes have been deposited in GISAID, but contributions from the United States, particularly through 2020, lagged behind the global effort. The primary goal of clinical microbiology laboratories is seldom rooted in epidemiologic or public health testing and many labs do not contain in-house sequencing technology. However, we recognized the need for clinical microbiologists to lend expertise, share specimen resources, and partner with academic laboratories and sequencing cores to assist in SARS-COV-2 epidemiologic sequencing efforts. Here we describe two clinical and academic laboratory collaborations for SARS-COV-2 genomic sequencing. We highlight roles of the clinical microbiologists and the academic labs, outline best practices, describe two divergent strategies in accomplishing a similar goal, and discuss the challenges with implementing and maintaining such programs

Orientia tsutsugamushi Modulates Endoplasmic Reticulum Stress to Benefit its Intracellular Growth and Targets NLRC5 to Inhibit Major Histocompatibility Complex I Expression

Scrub typhus, caused by the obligate intracellular bacterium Orientia tsutsugamushi, afflicts one million people annually. Despite being a global health threat, little is known about O. tsutsugamushi pathogenesis. Here, we demonstrate that O. tsutsugamushi modulates the ER and ER-associated processes as mechanisms of nutritional virulence and immune evasion. To obtain amino acids to fuel replication, O. tsutsugamushi simultaneously induces ER stress and the unfolded protein response (UPR) while inhibiting ER-associated degradation (ERAD) during early infection time points. During exponential growth, the bacterium releases the ER bottleneck, resulting in generation of ERAD-derived amino acids that it parasitized for replication. The O. tsutsugamushi effector, Ank4, is linked to this process, as it impedes ERAD when ectopically expressed. O. tsutsugamushi expression of ank4 peaks during the ERAD inhibition window, but is absent when the pathway is restored. These data reveal a novel mechanism of nutritional virulence, whereby an obligate intracellular pathogen coordinates the modulation of multiple ER-associated processes. Like other intracellular pathogens, O. tsutsugamushi inhibits expression of MHC-I, but it does so in a novel manner by degrading the master regulator of MHC-I, NLRC5. This impedes production of the MHC-I components, human leukocyte antigen A and Beta-2 microglobulin. The NLRC5-reduction mechanism recapitulates across diverse cell types, but the degree and duration of inhibition is cell type-specific. NLRC5 modulation and MHC-I inhibition are linked to another O. tsutsugamushi Ank, Ank5. NLRC5 is a putative interacting partner of Ank5. Moreover, NLRC5 and MHC-I levels are reduced in cells ectopically expressing Ank5. To our knowledge, these are the first examples of a pathogen modulating NLRC5 to negatively regulate MHC-I expression and of a bacterial effector interacting with NLRC5. As we learn more about the bacterium’s ability to regulate its host cell, a unifying theme has emerged: modulation of the ER and ER-associated pathways. These projects reveal two novel mechanisms of O. tsutsugamushi pathogenesis, strategies to acquire the amino acids needed for replication and to decrease MHC-I antigen presentation by the host cell. These insights help in understanding how O. tsutsugamushi and potentially other related pathogens co-opt host cell processes to cause disease

<i>Orientia tsutsugamushi</i> uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation

<div><p><i>Orientia tsutsugamushi</i> causes scrub typhus, a potentially fatal infection that threatens over one billion people. Nuclear translocation of the transcription factor, NF-κB, is the central initiating cellular event in the antimicrobial response. Here, we report that NF-κB p65 nuclear accumulation and NF-κB-dependent transcription are inhibited in <i>O</i>. <i>tsutsugamushi</i> infected HeLa cells and/or primary macrophages, even in the presence of TNFα. The bacterium modulates p65 subcellular localization by neither degrading it nor inhibiting IκBα degradation. Rather, it exploits host exportin 1 to mediate p65 nuclear export, as this phenomenon is leptomycin B-sensitive. <i>O</i>. <i>tsutsugamushi</i> antagonizes NF-κB-activated transcription even when exportin 1 is inhibited and NF-κB consequently remains in the nucleus. Two ankyrin repeat-containing effectors (Anks), Ank1 and Ank6, each of which possess a C-terminal F-box and exhibit 58.5% amino acid identity, are linked to the pathogen’s ability to modulate NF-κB. When ectopically expressed, both translocate to the nucleus, abrogate NF-κB-activated transcription in an exportin 1-independent manner, and pronouncedly reduce TNFα-induced p65 nuclear levels by exportin 1-dependent means. Flag-tagged Ank 1 and Ank6 co-immunoprecipitate p65 and exportin 1. Both also bind importin β1, a host protein that is essential for the classical nuclear import pathway. Importazole, which blocks importin β1 activity, abrogates Ank1 and Ank6 nuclear translocation. The Ank1 and Ank6 regions that bind importin β1 also mediate their transport into the nucleus. Yet, these regions are distinct from those that bind p65/exportin 1. The Ank1 and Ank6 F-box and the region that lies between it and the ankyrin repeat domain are essential for blocking p65 nuclear accumulation. These data reveal a novel mechanism by which <i>O</i>. <i>tsutsugamushi</i> modulates the activity and nuclear transport of NF-κB p65 and identify the first microbial proteins that co-opt both importin β1 and exportin 1 to antagonize a critical arm of the antimicrobial response.</p></div

Ank1 and Ank6 promote p65 removal from the nucleus in an exportin 1-dependent manner.

<p>HeLa cells were transfected to express Flag-tagged BAP, Ank1, Ank6, or IκBα SR. At 16 h, the cells were treated with LMB or vehicle control for 1 h. The media was replaced with media containing TNFα or vehicle for 30 min. The cells were then fixed, screened with antibodies specific for the Flag epitope and p65, and examined by confocal microscopy. Representative fluorescence images are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007023#ppat.1007023.s007" target="_blank">S7 Fig</a>. The mean percentage <u>+</u> SD of cells exhibiting p65 in the nucleus was determined. Quadruplicate samples of 100 cells each were counted per time point. Statistically significant (*<i>P</i> < 0.05; ***<i>P</i> < 0.001) values are indicated. n.s., not significant. Results are representative of three independent experiments.</p

Schematics of Ank1 and Ank6 mutant proteins.

<p>Schematics of full-length wild-type Ank1 and Ank6 are presented atop the left and right column, respectively, below each of which are schematics in which deleted (△) amino acids and corresponding domains are indicated. The amino termini consisting of residues 1 through 17 are represented by red boxes. Each annotated ankyrin repeat (AR) is denoted by a blue-filled arrow bordered by a solid line. The arrow filled with white dots over a blue background and bordered by a hatched line corresponds to the potential AR that is not annotated as an AR but exhibits 41.7% identity with Ank6 AR4. The yellow hexagon in Ank6 corresponds to a putative nuclear export sequence (NES) that is found in the C-terminal region of AR4. The corresponding amino acids of Ank1 (143–160) that align with the Ank6 putative NES but are not predicted to be an NES are denoted by a white hexagon. An orange box demarcates the intervening sequence region (ISR) corresponding to Ank1 residues 167 to 201 and Ank6 168 to 202. The C-terminal F-box of each Ank is indicated by a green box.</p

<i>O</i>. <i>tsutsugamushi</i> inhibits p65 nuclear accumulation.

<p>HeLa cells (A and B) or BMDMs (C and D) were infected with <i>O</i>. <i>tsutsugamushi</i> at an MOI of 1. At 2, 4, 8, 12, or 24 h, the cells were fixed and screened with antibodies against <i>O</i>. <i>tsutsugamushi</i> TSA56 (<i>Ot</i>) and p65 prior to examination by confocal microscopy. (A and C) Representative fluorescence images of cells viewed for <i>Ot</i>, p65, and merged images plus DAPI, which stains the nucleus, are presented. White arrows denote representative individual <i>O</i>. <i>tsutsugamushi</i> bacteria. (B and D) The mean percentage <u>+</u> SD of cells exhibiting p65 in the nucleus was determined at each time point. Triplicate samples of 100 cells each were counted per time point. Results are representative of three separate experiments.</p

Ank1 and Ank6 translocate into the nucleus and prevent p65 nuclear accumulation.

<p>(A) HeLa cells were transfected to express Flag-tagged BAP, Ank1, Ank6, or IκBα SR. At 16 h, the cells were exposed to TNFα or vehicle control for 30 min, after which they were fixed, screened with antibodies specific for the Flag epitope and p65, and examined by confocal microscopy. (B) The mean percentage <u>+</u> SD of transfected cells exhibiting p65 in the nucleus was determined. Triplicate samples of 100 cells each were counted per time point. Statistically significant (****<i>P</i> < 0.0001) values indicated. Results are representative of three independent experiments.</p

<i>O</i>. <i>tsutsugamushi</i> inhibits TNFα-stimulated p65 nuclear accumulation.

<p>HeLa cells (A and B) or BMDMs (C and D) were infected with <i>O</i>. <i>tsutsugamushi</i> at an MOI of 50 or 10, respectively, or mock infected. At 24, 48, or 72 h, the cells were treated with TNFα or vehicle control (Ctrl) for 30 min after which they were fixed, screened with antibodies against <i>O</i>. <i>tsutsugamushi</i> TSA56 (<i>Ot</i>) and p65, and visualized by confocal microscopy. (A and C) Representative fluorescence images of cells viewed for <i>Ot</i>, p65, and merged images plus DAPI, which stains the nucleus, are presented. (B and D) The mean percentage <u>+</u> SD of cells exhibiting p65 in the nucleus were determined at each time point. Triplicate samples of 100 cells each were counted per time point. Statistically significant (*<i>P</i> < 0.05; **<i>P</i><0.01; ***<i>P <</i> 0.001; ****<i>P</i> < 0.0001) values are indicated. n.s., not significant. Data are the mean <u>+</u> SD of three independent experiments performed in triplicate.</p