models-of-murine-vaginal-colonization-by-anaerobically-grown-bacteria

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...

Invasion of host cells by apicomplexan parasites such as  is critical for their infectivity and pathogenesis. In , secretion of essential egress, motility, and invasion-related proteins from microneme organelles is regulated by oscillations of intracellular Ca Later stages of invasion are considered Ca independent, including the secretion of proteins required for host cell entry and remodeling from the parasite's rhoptries. We identified a family of three  proteins with homology to the ferlin family of double C2 domain-containing Ca sensors. In humans and model organisms, such Ca sensors orchestrate Ca-dependent exocytic membrane fusion with the plasma membrane. Here we focus on one ferlin that is conserved across the ,  FER2 (TgFER2). Unexpectedly, conditionally TgFER2-depleted parasites secreted their micronemes normally and were completely motile. However, these parasites were unable to invade host cells and were therefore not viable. Knockdown of TgFER2 prevented rhoptry secretion, and these parasites failed to form the moving junction at the parasite-host interface necessary for host cell invasion. Collectively, these data demonstrate the requirement of TgFER2 for rhoptry secretion in  tachyzoites and suggest a possible Ca dependence of rhoptry secretion. These findings provide the first mechanistic insights into this critical yet poorly understood aspect of apicomplexan host cell invasion. Apicomplexan protozoan parasites, such as those causing malaria and toxoplasmosis, must invade the cells of their hosts in order to establish a pathogenic infection. Timely release of proteins from a series of apical organelles is required for invasion. Neither the vesicular fusion events that underlie secretion nor the observed reliance of the various processes on changes in intracellular calcium concentrations is completely understood. We identified a group of three proteins with strong homology to the calcium-sensing ferlin family, which are known to be involved in protein secretion in other organisms. Surprisingly, decreasing the amounts of one of these proteins (TgFER2) did not have any effect on the typically calcium-dependent steps in invasion. Instead, TgFER2 was essential for the release of proteins from organelles called rhoptries. These data provide a tantalizing first look at the mechanisms controlling the very poorly understood process of rhoptry secretion, which is essential for the parasite's infection cycle.

A Member of the Ferlin Calcium Sensor Family Is Essential for Toxoplasma gondii Rhoptry Secretion.

Sialylation of lacto--neotetraose (LNnT) extending from heptose I (HepI) of gonococcal lipooligosaccharide (LOS) contributes to pathogenesis. Previously, gonococcal LOS sialyltransterase (Lst) was shown to sialylate LOS in Triton X-100 extracts of strain 15253, which expresses lactose from both HepI and HepII, the minimal structure required for monoclonal antibody (MAb) 2C7 binding. Ongoing work has shown that growth of 15253 in cytidine monophospho--acetylneuraminic acid (CMP-Neu5Ac)-containing medium enables binding to CD33/Siglec-3, a cell surface receptor that binds sialic acid, suggesting that lactose termini on LOSs of intact gonococci can be sialylated. Neu5Ac was detected on LOSs of strains 15253 and an MS11 mutant with lactose only from HepI and HepII by mass spectrometry; deleting HepII lactose rendered Neu5Ac undetectable. Resistance of HepII lactose Neu5Ac to desialylation by α2-3-specific neuraminidase suggested an α2-6 linkage. Although not associated with increased factor H binding, HepII lactose sialylation inhibited complement C3 deposition on gonococci. Strain 15253 mutants that lacked Lst or HepII lactose were significantly attenuated in mice, confirming the importance of HepII Neu5Ac in virulence. All 75 minimally passaged clinical isolates from Nanjing, China, expressed HepII lactose, evidenced by reactivity with MAb 2C7; MAb 2C7 was bactericidal against the first 62 (of 75) isolates that had been collected sequentially and were sialylated before testing. MAb 2C7 effectively attenuated 15253 vaginal colonization in mice. In conclusion, this novel sialylation site could explain the ubiquity of gonococcal HepII lactose  Our findings reinforce the candidacy of the 2C7 epitope as a vaccine antigen and MAb 2C7 as an immunotherapeutic antibody.

A Novel Sialylation Site on Neisseria gonorrhoeae Lipooligosaccharide Links Heptose II Lactose Expression with Pathogenicity.

Natural killer (NK) cells are innate immune lymphocytes that recognize and destroy abnormal host cells, such as tumor cells or those infected by viral pathogens. To safely accomplish these functions, NK cells display activating receptors that detect stress molecules or viral ligands displayed at the cell surface, balanced by inhibitory receptors that bind to self-molecules. To date, such activating and inhibitory receptors on NK cells are not known to recognize bacterial determinants. Moreover, NK cell responses to direct interactions with extracellular bacteria are poorly explored. In this study, we observed the human neonatal pathogen group B  (GBS) can directly engage human NK cells. The interaction was mediated through the B6N segment of streptococcal β-protein, binding to the inhibitory receptor Siglec-7 via its amino-terminal V-set domain. Unlike classical Siglec binding, the interaction is also independent of its sialic acid recognition property. In contrast to WT GBS, mutants lacking β-protein induced efficient pyroptosis of NK cells through the NLRP3 inflammasome, with production and secretion of the proinflammatory cytokine IL-1β and dissemination of the cytotoxic molecule granzyme B. We postulate that GBS evolved β-protein engagement of inhibitory human Siglec-7 to suppress the pyroptotic response of NK cells and thereby block recruitment of a broader innate immune response, i.e., by "silencing the sentinel."

Siglec-7 engagement by GBS β-protein suppresses pyroptotic cell death of natural killer cells.

Controversies about the subcellular localization and mechanisms of action of the Alzheimer's disease-protective CD33 splice variant.

 deploys a unique immune evasion strategy wherein the lacto--neotetraose termini of lipooligosaccharide (LOS) are "capped" by a surface LOS sialyltransferase (Lst), using extracellular host-derived CMP-sialic acid (CMP-Neu5Ac in humans). LOS sialylation enhances complement resistance by recruiting factor H (FH; alternative complement pathway inhibitor) and also by limiting classical pathway activation. Sialylated LOS also engages inhibitory Siglecs on host leukocytes, dampening innate immunity. Previously, we showed that analogues of CMP-sialic acids (CMP-nonulosonates [CMP-NulOs]), such as CMP-Leg5,7Ac and CMP-Neu5Ac9N, are also substrates for Lst. Incorporation of Leg5,7Ac and Neu5Ac9N into LOS results in  being fully serum sensitive. Importantly, intravaginal administration of CMP-Leg5,7Ac attenuated  colonization of mouse vaginas. In this study, we characterize and develop additional candidate therapeutic CMP-NulOs. CMP-ketodeoxynonulosonate (CMP-Kdn) and CMP-Kdn7N, but not CMP-Neu4,5Ac, were substrates for Lst, further elucidating gonococcal Lst specificity. Lacto--neotetraose LOS capped with Kdn and Kdn7N bound FH to levels ∼60% of that seen with Neu5Ac and enabled gonococci to resist low (3.3%) but not higher (10%) concentrations of human complement. CMP-Kdn, CMP-Neu5Ac9N, and CMP-Leg5,7Ac administered intravaginally (10 μg/d) to -colonized mice were equally efficacious. Of the three CMP-NulOs above, CMP-Leg5,7Ac was the most pH and temperature stable. In addition, Leg5,7Ac-fed human cells did not display this NulO on their surface. Moreover, CMP-Leg5,7Ac was efficacious against several multidrug-resistant gonococci in mice with a humanized sialome ( mice) or humanized complement system (FH/C4b-binding protein transgenic mice). CMP-Leg5,7Ac and CMP-Kdn remain viable leads as topical preventive/therapeutic agents against the global threat of multidrug-resistant .

Therapeutic CMP-Nonulosonates against Multidrug-Resistant .

Novel therapies to counteract multidrug-resistant gonorrhea are urgently needed. A unique gonococcal immune evasion strategy involves capping of lipooligosaccharide (LOS) with sialic acid by gonococcal sialyltransferase (Lst), utilizing host-derived CMP-sialic acid (CMP-Neu5Ac in humans). LOS sialylation renders gonococci resistant to complement and cationic peptides, and down-regulates the inflammatory response by engaging siglecs. CMP-sialic acid analogs (CMP-nonulosonates [CMP-NulOs]) such as CMP-Leg5,7Ac2 and CMP-Kdn are also utilized by Lst. Incorporation of these NulO analogs into LOS maintains gonococci susceptible to complement. Intravaginal administration of CMP-Kdn or CMP-Leg5,7Ac2 attenuates gonococcal colonization of mouse vaginas. Here, we identify a key mechanism of action for the efficacy of CMP-NulOs. Surprisingly, CMP-NulOs remained effective in complement C1q-/- and C3-/- mice. LOS Neu5Ac, but not Leg5,7Ac2 or Kdn, conferred resistance to the cathelicidins LL-37 (human) and mouse cathelicidin-related antimicrobial peptide in vitro. CMP-NulOs were ineffective in Camp-/- mice, revealing that cathelicidins largely mediate the efficacy of therapeutic CMP-NulOs.

Efficacy of Antigonococcal CMP-Nonulosonate Therapeutics Require Cathelicidins.

Human metabolic incorporation of nonhuman sialic acid (Sia) N-glycolylneuraminic acid into endogenous glycans generates inflammation via preexisting antibodies, which likely contributes to red meat-induced atherosclerosis acceleration. Exploring whether this mechanism affects atherosclerosis in end-stage renal disease (ESRD), we instead found serum accumulation of 2-keto-3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (Kdn), a Sia prominently expressed in cold-blooded vertebrates. In patients with ESRD, levels of the Kdn precursor mannose also increased, but within a normal range. Mannose ingestion by healthy volunteers raised the levels of urinary mannose and Kdn. Kdn production pathways remained conserved in mammals but were diminished by an M42T substitution in a key biosynthetic enzyme, N-acetylneuraminate synthase. Remarkably, reversion to the ancestral methionine then occurred independently in 2 lineages, including humans. However, mammalian glycan databases contain no Kdn-glycans. We hypothesize that the potential toxicity of excess mannose in mammals is partly buffered by conversion to free Kdn. Thus, mammals probably conserve Kdn biosynthesis and modulate it in a lineage-specific manner, not for glycosylation, but to control physiological mannose intermediates and metabolites. However, human cells can be forced to express Kdn-glycans via genetic mutations enhancing Kdn utilization, or by transfection with fish enzymes producing cytidine monophosphate-Kdn (CMP-Kdn). Antibodies against Kdn-glycans occur in pooled human immunoglobulins. Pathological conditions that elevate Kdn levels could therefore result in antibody-mediated inflammatory pathologies.

Evolutionary conservation of human ketodeoxynonulosonic acid production is independent of sialoglycan biosynthesis.

Surface expression of the common vertebrate sialic acid (Sia) -acetylneuraminic acid (Neu5Ac) by commensal and pathogenic microbes appears structurally to represent "molecular mimicry" of host sialoglycans, facilitating multiple mechanisms of host immune evasion. In contrast, ketodeoxynonulosonic acid (Kdn) is a more ancestral Sia also present in prokaryotic glycoconjugates that are structurally quite distinct from vertebrate sialoglycans. We detected human antibodies against Kdn-terminated glycans, and sialoglycan microarray studies found these anti-Kdn antibodies to be directed against Kdn-sialoglycans structurally similar to those on human cell surface Neu5Ac-sialoglycans. Anti-Kdn-glycan antibodies appear during infancy in a pattern similar to those generated following incorporation of the nonhuman Sia -glycolylneuraminic acid (Neu5Gc) onto the surface of nontypeable  (NTHi), a human commensal and opportunistic pathogen. NTHi grown in the presence of free Kdn took up and incorporated the Sia into its lipooligosaccharide (LOS). Surface display of the Kdn within NTHi LOS blunted several virulence attributes of the pathogen, including Neu5Ac-mediated resistance to complement and whole blood killing, complement C3 deposition, IgM binding, and engagement of Siglec-9. Upper airway administration of Kdn reduced NTHi infection in human-like  null (Neu5Gc-deficient) mice that express a Neu5Ac-rich sialome. We propose a mechanism for the induction of anti-Kdn antibodies in humans, suggesting that Kdn could be a natural and/or therapeutic "Trojan horse" that impairs colonization and virulence phenotypes of free Neu5Ac-assimilating human pathogens. All cells in vertebrates are coated with a dense array of glycans often capped with sugars called sialic acids. Sialic acids have many functions, including serving as a signal for recognition of "self" cells by the immune system, thereby guiding an appropriate immune response against foreign "nonself" and/or damaged cells. Several pathogenic bacteria have evolved mechanisms to cloak themselves with sialic acids and evade immune responses. Here we explore a type of sialic acid called "Kdn" (ketodeoxynonulosonic acid) that has not received much attention in the past and compare and contrast how it interacts with the immune system. Our results show potential for the use of Kdn as a natural intervention against pathogenic bacteria that take up and coat themselves with external sialic acid from the environment.

Exploring the Impact of Ketodeoxynonulosonic Acid in Host-Pathogen Interactions Using Uptake and Surface Display by Nontypeable Haemophilus influenzae.

Article	Year
A Member of the Ferlin Calcium Sensor Family Is Essential for Toxoplasma gondii Rhoptry Secretion. mBio\| PubMed ID: 30279285	2018
A Novel Sialylation Site on Neisseria gonorrhoeae Lipooligosaccharide Links Heptose II Lactose Expression with Pathogenicity. Infection and immunity\| PubMed ID: 29844237	2018
Siglec-7 engagement by GBS β-protein suppresses pyroptotic cell death of natural killer cells. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 30254166	2018
Controversies about the subcellular localization and mechanisms of action of the Alzheimer's disease-protective CD33 splice variant. Acta neuropathologica\| PubMed ID: 31440824	2019
Therapeutic CMP-Nonulosonates against Multidrug-Resistant . Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 32434942	2020
Efficacy of Antigonococcal CMP-Nonulosonate Therapeutics Require Cathelicidins. The Journal of infectious diseases\| PubMed ID: 32692363	2020
Evolutionary conservation of human ketodeoxynonulosonic acid production is independent of sialoglycan biosynthesis. The Journal of clinical investigation\| PubMed ID: 33373330	2021
Exploring the Impact of Ketodeoxynonulosonic Acid in Host-Pathogen Interactions Using Uptake and Surface Display by Nontypeable Haemophilus influenzae. mBio\| PubMed ID: 33468699	2021

Sudeshna Saha

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