Annotation of Functions of Sequences of Concern and Its Relevance to the New Biosecurity Regulatory Framework in the United States.

Introduction: Recent regulations from United States Government agencies reshape the screening of synthetic nucleic acids. These take a step away from categorizing hazard on the basis of "bad" taxa and invoke the function of the sequence in pathogenesis or intoxication. Ascertaining functions related to pathogenesis and distinguishing these from other molecular abilities that are unproblematic is not simple. Some have suggested that this information can be readily obtained from existing databases of pathogens. Objectives: We evaluate how virulence factors are described in current databases of pathogens and their adequacy for biothreat data science. We discuss limitations of how virulence factors have been conceived and propose using the sequence of concern (SoC) term to distinguish sequences with biothreat from those without. We discuss ways in which databases of SoCs might be implemented for research and regulatory purposes. We describe ongoing work improving functional descriptions of SoCs. Methods: We assess the adequacy of descriptions of virulence factors in pathogen databases following extensive engagement with the literature in microbial pathogenesis. Results/Conclusions: Descriptions of virulence factors in pathogen databases are inadequate for understanding biothreats. Many are not biothreats and would not be concerning if transferred to another pathogen. New gene ontology terms have been authored, and those specific to pathogenic viral processes are being generalized to make them relevant to other pathogenic taxa. This allows better understanding by humans and better recognition by machines. A database of annotated functions of SoCs could benefit the evolving biosecurity regulatory framework in the United States.

Lyme Neuroborreliosis in the Context of Dementia Syndromes.

Lyme disease (LD) can affect the skin, joints, heart, and nervous system as a multisystemic condition. The cause of the illness is the spirochete of the genus Borrelia. These pathogens can affect the skin, joints, heart, and nervous system. Lyme neuroborreliosis (LNB) is the term for the disease, which occurs when the nervous system gets involved. Regarding geographical distribution, LNB is more prevalent in Europe than in North America. The most significant change in pathogenesis is inflammation of the central nervous system (CNS) and peripheral nervous system (PNS). Furthermore, clinically, it can represent a variety of neurological manifestations, such as meningitis, encephalitis, radiculopathies, and cranial neuritis. However, dementia-like syndrome is an infrequent manifestation of Lyme disease. Our review article aims to summarize the similarities and differences between dementia-like syndrome in LNB and that in primary neurodegenerative diseases, as well as to look for a correlation between the pathogenesis of the disease and the possibility of developing dementia-like syndrome. The world literature lacks sufficiently convincing data on the relationship between spirochete infection and primary dementia syndromes. However, cases of secondary dementia syndrome due to nervous system involvement as well as post-treatment have been described. A thorough examination, medical history, laboratory and imaging studies, cerebrospinal fluid (CSF) examination, MRI, and fludeoxyglucose-18-positron emission tomography (FGD-PET) are required to differentiate between these syndromes.

RNA:DNA triplexes: a mechanism for epigenetic communication between hosts and microbes?

Molecular communication between host and microbe is mediated by the transfer of many different classes of macromolecules. Recently, the trafficking of RNA molecules between organisms has gained prominence as an efficient way to manipulate gene expression via RNA interference (RNAi). Here, we posit a new epigenetic control mechanism based on triple helix (triplex) structures comprising nucleic acids from both host and microbe. Indeed, RNA:DNA triplexes are known to regulate gene expression in humans, but it is unknown whether interkingdom triplexes are formed either to manipulate host processes during pathogenesis or as a host defense response. We hypothesize that a fraction of the extracellular RNAs commonly released by microbes (e.g., bacteria, fungi, and protists) and their hosts form triplexes with the genome of the other species, thereby impacting chromatin conformation and gene expression. We invite the field to consider interkingdom triplexes as unexplored weaponry in the arms race between host and microbe.

Translocon Subunits of the COP9 Signalosome Complex Are a Central Hub for Regulating Multiple Photoresponsive Processes and Autophagic Flux in Magnaporthe oryzae.

Photodependent processes, including circadian rhythm, autophagy, ubiquitination, neddylation/deneddylation, and metabolite biosynthesis, profoundly influence microbial pathogenesis. Although a photomorphogenesis signalosome (COP9/CSN) has been identified, the mechanism by which this large complex contributes to the pathophysiological processes in filamentous fungi remains unclear. Here, we identified eight CSN complex subunits in the rice blast fungus Magnaporthe oryzae and functionally characterized the translocon subunits containing a nuclear export or localization signal (NES/NLS). Targeted gene replacement of these CSN subunits, including MoCSN3, MoCSN5, MoCSN6, MoCSN7, and MoCSN12, attenuated vegetative growth and conidiation and rendered the deletion strains nonpathogenic. MoCSN7 deletion significantly suppressed arachidonic acid catabolism, and compromised cell wall integrity in M. oryzae. Surprisingly, we also discovered that MoCSN subunits, particularly MoCsn7, are required for the cAMP-dependent regulation of autophagic flux. Therefore, MoCSN significantly contributes to morphological, physiological, and pathogenic differentiation in M. oryzae by fostering cross-talk between multiple pathways.

The Chlamydia effector IncE employs two short linear motifs to reprogram host vesicle trafficking.

Chlamydia trachomatis, a leading cause of bacterial sexually transmitted infections, creates a specialized intracellular replicative niche by translocation and insertion of a diverse array of effectors (Incs [inclusion membrane proteins]) into the inclusion membrane. Here, we characterize IncE, a multifunctional Inc that encodes two non-overlapping short linear motifs (SLiMs) within its short cytosolic C terminus. The proximal SLiM, by mimicking just a small portion of an R-N-ethylmaleimide-sensitive factor adaptor protein receptor (SNARE) motif, binds and recruits syntaxin (STX)7- and STX12-containing vesicles to the inclusion. The distal SLiM mimics the sorting nexin (SNX)5 and SNX6 cargo binding site to recruit SNX6-containing vesicles to the inclusion. By simultaneously binding two distinct vesicle classes, IncE brings these vesicles in close apposition with each other at the inclusion to facilitate C. trachomatis intracellular development. Our work suggests that Incs may have evolved SLiMs to enable rapid evolution in a limited protein space to disrupt host cell processes.

Inhibition of host 5-lipoxygenase reduces overexuberant inflammatory responses and mortality associated with Cryptococcus meningoencephalitis.

Cryptococcosis, caused by fungi of the genus Cryptococcus, manifests in a broad range of clinical presentations, including severe pneumonia and disease of the central nervous system (CNS) and other tissues (bone and skin). Immune deficiency or development of overexuberant inflammatory responses can result in increased susceptibility or host damage, respectively, during fungal encounters. Leukotrienes help regulate inflammatory responses against fungal infections. Nevertheless, studies showed that Cryptococcus exploits host 5-lipoxygenase (5-LO), an enzyme central to the metabolism of arachidonic acid into leukotrienes, to facilitate transmigration across the brain-blood barrier. To investigate the impact of host 5-LO on the development of protective host immune responses and mortality during cryptococcosis, wild-type (C57BL/6) and 5-lipoxygenase-deficient (5-LO-/-) mice were given experimental pulmonary and systemic Cryptococcus sp., infections. Our results showed that 5-LO-/- mice exhibited reduced pathology and better disease outcomes (i.e., no mortality or signs associated with cryptococcal meningoencephalitis) following pulmonary infection with C. deneoformans, despite having detectable yeast in the brain tissues. In contrast, C57BL/6 mice exhibited classical signs associated with cryptococcal meningoencephalitis. Additionally, brain tissues of 5-LO-/- mice exhibited lower levels of cytokines (CCL2 and CCL3) clinically associated with Cryptococcus-related immune reconstitution inflammatory syndrome (C-IRIS). In a systemic mouse model of cryptococcosis, 5-LO-/- mice and those treated with a Federal Drug Administration (FDA)-approved 5-LO synthesis inhibitor, zileuton, displayed significantly reduced mortality compared to C57BL/6 infected mice. These results suggest that therapeutics designed to inhibit host 5-LO signaling could reduce disease pathology and mortality associated with cryptococcal meningoencephalitis. IMPORTANCE: Cryptococcosis is a mycosis with worldwide distribution and has a broad range of clinical manifestations, including diseases of the CNS. Globally, there is an estimated 179,000 cases of cryptococcal meningitis, resulting in approximately 112,000 fatalities per annum and 19% of AIDS-related deaths. Understanding how host immune responses are modulated during cryptococcosis is central to mitigating the morbidity and mortality associated with cryptococcosis. Leukotrienes (LTs) have been shown to modulate inflammatory responses during infection. In this study, we show that mice deficient in 5-lipoxygenase (5-LO), an enzyme central to the metabolism of arachidonic acid into leukotrienes, exhibit reduced pathology, disease, and neurological signs associated with cryptococcal meningitis. Additionally, mice given an experimental cryptococcal infection and subsequently treated with an FDA-approved 5-LO synthesis inhibitor exhibited significantly reduced mortality rates. These results suggest that therapeutics designed to inhibit host 5-LO activity could significantly reduce pathology and mortality rates associated with cryptococcal meningitis.

Advances in bioinspired nanomaterials managing microbial biofilms and virulence: A critical analysis.

Microbial virulence and biofilm formation stand as a big concern against the goal of achieving a green and sustainable future. Microbial pathogenesis is the process by which the microbes (bacterial, fungal, and viral) cause illness in their respective host organism. 'Nanotechnology' is a state-of-art discipline to address this problem. The use of conventional techniques against microbial proliferation has been challenging against the environment. To tackle this problem, there has been a revolution in this multi-disciplinary field, to address the aspect of bioinspired nanomaterials in the antibiofilm and antimicrobial sector. Bioinspired nanomaterials prove to be a potential antibiofilm and antimicrobial agent as they are non-hazardous to the environment and mostly synthesized using a single-step reduction protocol. They exhibit synergistic effects against bacterial, fungal, and viral pathogens and thereby, control the virulence. In this literature review, we have elucidated the potential of bioinspired nanoparticles as well as nanomaterials as a promising anti-microbial treatment pedagogy and throw light on the advancements in how smart photo-switchable platforms have been designed to exhibit both bacterial releasing as well as bacterial-killing properties. Certain limitations and possible outcomes of these bio-based nanomaterials have been discussed in the hope of achieving a green and sustainable ecosystem.

Draft genome sequences of a blaCTX-M-15-harboring and an atypical enteropathogenic Escherichia coli isolate recovered from slaughterhouses in Nigeria.

We present the draft genome sequences of two Escherichia coli strains isolated from slaughterhouses in Edo State, Nigeria, in 2019. The isolates were identified as blaCTX-M-15-harboring (19-47-58) and atypical enteropathogenic E. coli (aEPEC) (19-47-66), belonging to multilocus sequence types (MLST) ST46 and ST2089, respectively.

Synthesis, characterization, and applications of iron oxide nanoparticles.

Objective: The green synthesis method for nanoparticles is getting more attention globally, due to its lesser cost, non-hazardous, and eco-friendly nature. The novelty of the present work is to investigate the anti-bacterial and degradation activity of the green synthesized Iron Oxide NPs. Methods: In this study, the Iron Oxide NPs were synthesized through a green synthesis route from leaves of Ficus Palmata. UV-Vis confirmed Iron Oxide NP's peaks between (230-290 nm), while Fourier transforms infrared spectroscopy analysis showed that several groups were involved in reduction and stabilization. Results: Results indicated that the highest photo thermal activity was shown in light and it was almost 4 folds greater than the control. Similarly, Iron Oxide NPs showed excellent antimicrobial potential against bacterial species "Salmonella typhi" "Xanthomonas Oryzae" and "Lactobacillus" at low concentrations (150 µg/mL). Hemolytic assay results showed that the toxicity was lesser than 5% at both dark and light conditions. Moreover, we also evaluated the photo-catalytic potential of Iron Oxide NPs against methylene orange. Results indicated that almost complete degradation was noted after 90 min in the presence of continuous light. All tests were performed in triplicates. All the data was subjected to P-test (P < 0.5) using Excel and graph pad (V.5.0). Conclusion: Iron Oxide NPs holds a promising future and could be used in treating diseases, and microbial pathogenesis and also could be used as a vector in drug delivery. Moreover, they can also eradicate persistent dyes and could be used as an alternative to remediate pollutants from the environment.

L-arginine metabolism as pivotal interface of mutual host-microbe interactions in the gut.

L-arginine (L-arg) is a versatile amino acid and a central intestinal metabolite in mammalian and microbial organisms. Thus, L-arg participates as precursor of multiple metabolic pathways in the regulation of cell division and growth. It also serves as a source of carbon, nitrogen, and energy or as a substrate for protein synthesis. Consequently, L-arg can simultaneously modify mammalian immune functions, intraluminal metabolism, intestinal microbiota, and microbial pathogenesis. While dietary intake, protein turnover or de novo synthesis usually supply L-arg in sufficient amounts, the expression of several key enzymes of L-arg metabolism can change rapidly and dramatically following inflammation, sepsis, or injury. Consequently, the availability of L-arg can be restricted due to increased catabolism, transforming L-arg into an essential amino acid. Here, we review the enzymatic pathways of L-arg metabolism in microbial and mammalian cells and their role in immune function, intraluminal metabolism, colonization resistance, and microbial pathogenesis in the gut.

Pathogen protein modularity enables elaborate mimicry of a host phosphatase.

Pathogens produce diverse effector proteins to manipulate host cellular processes. However, how functional diversity is generated in an effector repertoire is poorly understood. Many effectors in the devastating plant pathogen Phytophthora contain tandem repeats of the "(L)WY" motif, which are structurally conserved but variable in sequences. Here, we discovered a functional module formed by a specific (L)WY-LWY combination in multiple Phytophthora effectors, which efficiently recruits the serine/threonine protein phosphatase 2A (PP2A) core enzyme in plant hosts. Crystal structure of an effector-PP2A complex shows that the (L)WY-LWY module enables hijacking of the host PP2A core enzyme to form functional holoenzymes. While sharing the PP2A-interacting module at the amino terminus, these effectors possess divergent C-terminal LWY units and regulate distinct sets of phosphoproteins in the host. Our results highlight the appropriation of an essential host phosphatase through molecular mimicry by pathogens and diversification promoted by protein modularity in an effector repertoire.

The Two Faces of Bacterial Membrane Vesicles: Pathophysiological Roles and Therapeutic Opportunities.

Bacterial membrane vesicles (MVs) are nanosized lipid particles secreted by lysis or blebbing mechanisms from Gram-negative and -positive bacteria. It is becoming increasingly evident that MVs can promote antimicrobial resistance but also provide versatile opportunities for therapeutic exploitation. As non-living facsimiles of parent bacteria, MVs can carry multiple bioactive molecules such as proteins, lipids, nucleic acids, and metabolites, which enable them to participate in intra- and interspecific communication. Although energetically costly, the release of MVs seems beneficial for bacterial fitness, especially for pathogens. In this review, we briefly discuss the current understanding of diverse MV biogenesis routes affecting MV cargo. We comprehensively highlight the physiological functions of MVs derived from human pathogens covering in vivo adaptation, colonization fitness, and effector delivery. Emphasis is given to recent findings suggesting a vicious cycle of MV biogenesis, pathophysiological function, and antibiotic therapy. We also summarize potential therapeutical applications, such as immunotherapy, vaccination, targeted delivery, and antimicrobial potency, including their experimental validation. This comparative overview identifies common and unique strategies for MV modification used along diverse applications. Thus, the review summarizes timely aspects of MV biology in a so far unprecedented combination ranging from beneficial function for bacterial pathogen survival to future medical applications.

Improved understanding of biorisk for research involving microbial modification using annotated sequences of concern.

Regulation of research on microbes that cause disease in humans has historically been focused on taxonomic lists of 'bad bugs'. However, given our increased knowledge of these pathogens through inexpensive genome sequencing, 5 decades of research in microbial pathogenesis, and the burgeoning capacity of synthetic biologists, the limitations of this approach are apparent. With heightened scientific and public attention focused on biosafety and biosecurity, and an ongoing review by US authorities of dual-use research oversight, this article proposes the incorporation of sequences of concern (SoCs) into the biorisk management regime governing genetic engineering of pathogens. SoCs enable pathogenesis in all microbes infecting hosts that are 'of concern' to human civilization. Here we review the functions of SoCs (FunSoCs) and discuss how they might bring clarity to potentially problematic research outcomes involving infectious agents. We believe that annotation of SoCs with FunSoCs has the potential to improve the likelihood that dual use research of concern is recognized by both scientists and regulators before it occurs.

Effector Identification in Plant Pathogens.

Effectors play a central role in determining the outcome of plant-pathogen interactions. As key virulence proteins, effectors are collectively indispensable for disease development. By understanding the virulence mechanisms of effectors, fundamental knowledge of microbial pathogenesis and disease resistance have been revealed. Effectors are also considered double-edged swords because some of them activate immunity in disease resistant plants after being recognized by specific immune receptors, which evolved to monitor pathogen presence or activity. Characterization of effector recognition by their cognate immune receptors and the downstream immune signaling pathways is instrumental in implementing resistance. Over the past decades, substantial research effort has focused on effector biology, especially concerning their interactions with virulence targets or immune receptors in plant cells. A foundation of this research is robust identification of the effector repertoire from a given pathogen, which depends heavily on bioinformatic prediction. In this review, we summarize methodologies that have been used for effector mining in various microbial pathogens which use different effector delivery mechanisms. We also discuss current limitations and provide perspectives on how recently developed analytic tools and technologies may facilitate effector identification and hence generation of a more complete vision of host-pathogen interactions. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Microbial Pathogenesis in the Era of Spatial Omics.

The biology of a cell, whether it is a unicellular organism or part of a multicellular network, is influenced by cell type, temporal changes in cell state, and the cell's environment. Spatial cues play a critical role in the regulation of microbial pathogenesis strategies. Information about where the pathogen is-in a tissue or in proximity to a host cell-regulates gene expression and the compartmentalization of gene products in the microbe and the host. Our understanding of host and pathogen identity has bloomed with the accessibility of transcriptomics and proteomics techniques. A missing piece of the puzzle has been our ability to evaluate global transcript and protein expression in the context of the subcellular niche, primary cell, or native tissue environment during infection. This barrier is now lower with the advent of new spatial omics techniques to understand how location regulates cellular functions. This review will discuss how recent advances in spatial proteomics and transcriptomics approaches can address outstanding questions in microbial pathogenesis.

Cryptococcus neoformans Mar1 function links mitochondrial metabolism, oxidative stress, and antifungal tolerance.

Introduction: Microbial pathogens undergo significant physiological changes during interactions with the infected host, including alterations in metabolism and cell architecture. The Cryptococcus neoformans Mar1 protein is required for the proper ordering of the fungal cell wall in response to host-relevant stresses. However, the precise mechanism by which this Cryptococcus-specific protein regulates cell wall homeostasis was not defined. Methods: Here, we use comparative transcriptomics, protein localization, and phenotypic analysis of a mar1D loss-of-function mutant strain to further define the role of C. neoformans Mar1 in stress response and antifungal resistance. Results: We demonstrate that C. neoformans Mar1 is highly enriched in mitochondria. Furthermore, a mar1Δ mutant strain is impaired in growth in the presence of select electron transport chain inhibitors, has altered ATP homeostasis, and promotes proper mitochondrial morphogenesis. Pharmacological inhibition of complex IV of the electron transport chain in wild-type cells promotes similar cell wall changes as the mar1Δ mutant strain, supporting prior associations between mitochondrial function and cell wall homeostasis. Although Mar1 is not required for general susceptibility to the azole antifungals, the mar1Δ mutant strain displays increased tolerance to fluconazole that correlates with repressed mitochondrial metabolic activity. Discussion: Together, these studies support an emerging model in which the metabolic activity of microbial cells directs cell physiological changes to allow persistence in the face of antimicrobial and host stress.

Extracellular vesicles in bacterial and fungal diseases - Pathogenesis to diagnostic biomarkers.

Intercellular communication among microbes plays an important role in disease exacerbation. Recent advances have described small vesicles, termed as "extracellular vesicles" (EVs), previously disregarded as "cellular dust" to be vital in the intracellular and intercellular communication in host-microbe interactions. These signals have been known to initiate host damage and transfer of a variety of cargo including proteins, lipid particles, DNA, mRNA, and miRNAs. Microbial EVs, referred to generally as "membrane vesicles" (MVs), play a key role in disease exacerbation suggesting their importance in pathogenicity. Host EVs help coordinate antimicrobial responses and prime the immune cells for pathogen attack. Hence EVs with their central role in microbe-host communication, may serve as important diagnostic biomarkers of microbial pathogenesis. In this review, we summarize current research regarding the roles of EVs as markers of microbial pathogenesis with specific focus on their interaction with host immune defence and their potential as diagnostic biomarkers in disease conditions.

AICAR transformylase/IMP cyclohydrolase (ATIC) is essential for de novo purine biosynthesis and infection by Cryptococcus neoformans.

The fungal pathogen Cryptococcus neoformans is a leading cause of meningoencephalitis in the immunocompromised. As current antifungal treatments are toxic to the host, costly, limited in their efficacy, and associated with drug resistance, there is an urgent need to identify vulnerabilities in fungal physiology to accelerate antifungal discovery efforts. Rational drug design was pioneered in de novo purine biosynthesis as the end products of the pathway, ATP and GTP, are essential for replication, transcription, and energy metabolism, and the same rationale applies when considering the pathway as an antifungal target. Here, we describe the identification and characterization of C. neoformans 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/5'-inosine monophosphate cyclohydrolase (ATIC), a bifunctional enzyme that catalyzes the final two enzymatic steps in the formation of the first purine base inosine monophosphate. We demonstrate that mutants lacking the ATIC-encoding ADE16 gene are adenine and histidine auxotrophs that are unable to establish an infection in a murine model of virulence. In addition, our assays employing recombinantly expressed and purified C. neoformans ATIC enzyme revealed Km values for its substrates AICAR and 5-formyl-AICAR are 8-fold and 20-fold higher, respectively, than in the human ortholog. Subsequently, we performed crystallographic studies that enabled the determination of the first fungal ATIC protein structure, revealing a key serine-to-tyrosine substitution in the active site, which has the potential to assist the design of fungus-specific inhibitors. Overall, our results validate ATIC as a promising antifungal drug target.

The SET-domain protein CgSet4 negatively regulates antifungal drug resistance via the ergosterol biosynthesis transcriptional regulator CgUpc2a.

Invasive fungal infections, which pose a serious threat to human health, are increasingly associated with a high mortality rate and elevated health care costs, owing to rising resistance to current antifungals and emergence of multidrug-resistant fungal species. Candida glabrata is the second to fourth common cause of Candida bloodstream infections. Its high propensity to acquire resistance toward two mainstream drugs, azoles (inhibit ergosterol biosynthesis) and echinocandins (target cell wall), in clinical settings, and its inherent low azole susceptibility render antifungal therapy unsuccessful in many cases. Here, we demonstrate a pivotal role for the SET {suppressor of variegation 3 to 9 [Su(var)3-9], enhancer of zeste [E(z)], and trithorax (Trx)} domain-containing protein, CgSet4, in azole and echinocandin resistance via negative regulation of multidrug transporter-encoding and ergosterol biosynthesis (ERG) genes through the master transcriptional factors CgPdr1 and CgUpc2A, respectively. RNA-Seq analysis revealed that C. glabrata responds to caspofungin (CSP; echinocandin antifungal) stress by downregulation and upregulation of ERG and cell wall organization genes, respectively. Although CgSet4 acts as a repressor of the ergosterol biosynthesis pathway via CgUPC2A transcriptional downregulation, the CSP-induced ERG gene repression is not dependent on CgSet4, as CgSet4 showed diminished abundance on the CgUPC2A promoter in CSP-treated cells. Furthermore, we show a role for the last three enzymes of the ergosterol biosynthesis pathway, CgErg3, CgErg5, and CgErg4, in antifungal susceptibility and virulence in C. glabrata. Altogether, our results unveil the link between ergosterol biosynthesis and echinocandin resistance and have implications for combination antifungal therapy.