Brianna Dalesandro

Front Microbiol. 2022 Aug 9;13:986396. doi: 10.3389/fmicb.2022.986396. eCollection 2022.

ABSTRACT

Selected lactic acid bacteria can stimulate macrophages and dendritic cells to secrete IL-12, which plays a key role in activating innate and cellular immunity. In this study, we investigated the roles of cell wall teichoic acids (WTAs) displayed on whole intact cell walls (ICWs) of Lactiplantibacillus plantarum in activation of mouse macrophages. ICWs were prepared from whole bacterial cells of several lactobacilli without physical disruption, and thus retaining the overall shapes of the bacteria. WTA-displaying ICWs of several L. plantarum strains, but not WTA-lacking ICWs of strains of other lactobacilli, elicited IL-12 secretion from mouse bone marrow-derived macrophages (BMMs) and mouse macrophage-like J774.1 cells. The ability of the ICWs of L. plantarum to induce IL-12 secretion was abolished by selective chemical elimination of WTAs from ICWs, but was preserved by selective removal of cell wall glycopolymers other than WTAs. BMMs prepared from TLR2- or TLR4-deficient mouse could secret IL-12 upon stimulation with ICWs of L. plantarum and a MyD88 dimerization inhibitor did not affect ICW-mediated IL-12 secretion. WTA-displaying ICWs, but not WTA-lacking ICWs, were ingested in the cells within 30 min. Treatment with inhibitors of actin polymerization abolished IL-12 secretion in response to ICW stimulation and diminished ingestion of ICWs. When overall shapes of ICWs of L. plantarum were physically disrupted, the disrupted ICWs (DCWs) failed to induce IL-12 secretion. However, DCWs and soluble WTAs inhibited ICW-mediated IL-12 secretion from macrophages. Taken together, these results show that WTA-displaying ICWs of L. plantarum can elicit IL-12 production from macrophages via actin-dependent phagocytosis but TLR2 signaling axis independent pathway. WTAs displayed on ICWs are key molecules in the elicitation of IL-12 secretion, and the sizes and shapes of the ICWs have an impact on actin remodeling and subsequent IL-12 production.

PMID:36016797 | PMC:PMC9396385 | DOI:10.3389/fmicb.2022.986396

J Immunol. 2022 Sep 15;209(6):1146-1155. doi: 10.4049/jimmunol.2200080. Epub 2022 Aug 24.

ABSTRACT

IgG molecules are crucial for the human immune response against bacterial infections. IgGs can trigger phagocytosis by innate immune cells, like neutrophils. To do so, IgGs should bind to the bacterial surface via their variable Fab regions and interact with Fcγ receptors and complement C1 via the constant Fc domain. C1 binding to IgG-labeled bacteria activates the complement cascade, which results in bacterial decoration with C3-derived molecules that are recognized by complement receptors on neutrophils. Next to FcγRs and complement receptors on the membrane, neutrophils also express the intracellular neonatal Fc receptor (FcRn). We previously reported that staphylococcal protein A (SpA), a key immune-evasion protein of Staphylococcus aureus, potently blocks IgG-mediated complement activation and killing of S. aureus by interfering with IgG hexamer formation. SpA is also known to block IgG-mediated phagocytosis in absence of complement, but the mechanism behind it remains unclear. In this study, we demonstrate that SpA blocks IgG-mediated phagocytosis and killing of S. aureus and that it inhibits the interaction of IgGs with FcγRs (FcγRIIa and FcγRIIIb, but not FcγRI) and FcRn. Furthermore, our data show that multiple SpA domains are needed to effectively block IgG1-mediated phagocytosis. This provides a rationale for the fact that SpA from S. aureus contains four to five repeats. Taken together, our study elucidates the molecular mechanism by which SpA blocks IgG-mediated phagocytosis and supports the idea that in addition to FcγRs, the intracellular FcRn is also prevented from binding IgG by SpA.

PMID:36002230 | DOI:10.4049/jimmunol.2200080

Nat Commun. 2022 Aug 8;13(1):4614. doi: 10.1038/s41467-022-32423-9.

ABSTRACT

Single-chain variable fragments (scFvs), composed of variable domains of heavy and light chains of an antibody joined by a linker, share antigen binding capacity with their parental antibody. Due to intrinsically low solubility and stability, only two Escherichia coli-produced scFvs have been approved for therapy. Here we report that a 33-residue peptide, termed P17 tag, increases the solubility of multiple scFvs produced in Escherichia coli SHuffle strain by up to 11.6 fold. Hydrophilic sequence, especially charged residues, but not the predicted α-helical secondary structure of P17 tag, contribute to the solubility enhancement. Notably, the P17 tag elevates the thermostability of scFv as efficiently as intra-domain disulfide bonds. Moreover, a P17-tagged scFv targeting hepatitis B virus surface proteins shows over two-fold higher antigen-binding affinity and virus-neutralizing activity than the untagged version. These data strongly suggest a type I intramolecular chaperone-like activity of the P17 tag. Hence, the P17 tag could benefit the research, production, and application of scFv.

PMID:35941164 | PMC:PMC9359998 | DOI:10.1038/s41467-022-32423-9

APMIS. 2022 Sep;130(9):578-589. doi: 10.1111/apm.13258. Epub 2022 Jul 12.

ABSTRACT

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to almost all β-lactam antibiotics. Hence, new ways to control MRSA infection, such as antibacterial antibodies, need to be explored. α-hemolysin is the most important virulence factor widely expressed in S. aureus. This study aimed to develop a new fully human antibody against α-hemolysin of S. aureus and research its neutralizing effect. The single-chain antibody fragments (scFvs) against S. aureus were screened from a fully human scFv library using phage display technology. The selected scFvs had good binding affinities to α-hemolysin and S. aureus. The IgG-like scFv-Fc inserted into the pcDNA3.1 or pMH3 vector was expressed in HEK293F suspension cells to extend the half-life and restore Fc function. The size of purified scFv-Fc was about 55 kDa. The functions of expressed scFv-Fcs against α-hemolysin were validated. The cytotoxicity assays showed that scFv555-Fc had better protective effects on A549 cells than other scFv-Fcs. The results of anti-rabbit erythrocyte lysis and A549 cell apoptosis assay confirmed that scFv555-Fc had a significant neutralizing effect on α-hemolysin. The scFv555-Fc was used to construct the docking model of antigen-antibody complexes using Discovery Studio software. It predicted that the key binding sites of α-hemolysin were TYR28, LYS37, PHE39, ARG56, and LYS58, which might be the key toxic sites of α-hemolysin. A novel fully human scFv-Fc antibody neutralizing the α-hemolysin toxin of S. aureus was successfully developed. The findings might provide a new theoretical basis and treatment method for preventing MRSA infection.

PMID:35751523 | DOI:10.1111/apm.13258

Mol Immunol. 2022 Oct;150:47-57. doi: 10.1016/j.molimm.2022.08.005. Epub 2022 Aug 17.

ABSTRACT

The increasing incidence reports of antibiotic resistance highlights the need for alternative approaches to deal with bacterial infections. This brought about the idea of utilizing monoclonal antibodies as an alternative antibacterial treatment. Majority of the studies are focused on developing antibodies to bacterial surface antigens, with little emphasis on antibodies that inhibit the growth mechanisms of a bacteria host. Isocitrate lyase (ICL) is an important enzyme for the growth and survival of Mycobacterium tuberculosis (MTB) during latent infection as a result of its involvement in the mycobacterial glyoxylate and methylisocitrate cycles. It is postulated that the inhibition of ICL can disrupt the life cycle of MTB. To this extent, we utilized antibody phage display to identify a single chain fragment variable (scFv) antibody against the recombinant ICL protein from MTB. The soluble a-ICL-C6 scFv clone exhibited good binding characteristics with high specificity against ICL. More importantly, the clone exhibited in vitro inhibitory effect with an enzymatic assay resulting in a decrease of ICL enzymatic activity. In silico analysis showed that the scFv-ICL interactions are driven by 23 hydrogen bonds and 13 salt bridges that might disrupt the formation of ICL subunits for the tertiary structure or the formation of active site β domain. However, further validation is necessary to confirm if the isolated clone is indeed a good inhibitor against ICL for application against MTB.

PMID:35987135 | DOI:10.1016/j.molimm.2022.08.005

Nat Commun. 2022 Aug 2;13(1):4468. doi: 10.1038/s41467-022-31932-x.

ABSTRACT

Bacteria-based tumor therapy has recently attracted wide attentions due to its unique capability in targeting tumors and preferentially colonizing the core area of the tumor. Various therapeutic genes are also harbored into these engineering bacteria to enhance their anti-tumor efficacy. However, it is difficult to spatiotemporally control the expression of these inserted genes in the tumor site. Here, we engineer an ultrasound-responsive bacterium (URB) which can induce the expression of exogenous genes in an ultrasound-controllable manner. Owing to the advantage of ultrasound in tissue penetration, an acoustic remote control of bacterial gene expression can be realized by designing a temperature-actuated genetic switch. Cytokine interferon-γ (IFN-γ), an important immune regulatory molecule that plays a significant role in tumor immunotherapy, is used to test the system. Our results show that brief hyperthermia induced by focused ultrasound promotes the expression of IFN-γ gene, improving anti-tumor efficacy of URB in vitro and in vivo. Our study provides an alternative strategy for bacteria-mediated tumor immunotherapy.

PMID:35918309 | PMC:PMC9345953 | DOI:10.1038/s41467-022-31932-x

ACS Infect Dis. 2022 Sep 9;8(9):1831-1838. doi: 10.1021/acsinfecdis.2c00333. Epub 2022 Aug 4.

ABSTRACT

The human oral microbiome is the second largest microbial community in humans, harboring over 700 bacterial species, which aid in digestion and protect from growth of disease-causing pathogens. One such oral pathogen, Tannerella forsythia, along with other species, contributes to the pathogenesis of periodontitis. T. forsythia is unable to produce its own N-acetylmuramic acid (NAM) sugar, essential for peptidoglycan biosynthesis and therefore must scavenge NAM from other species with which it cohabitates. Here, we explore the recycling potential of T. forsythia for NAM uptake with a bioorthogonal modification into its peptidoglycan, allowing for click-chemistry-based visualization of the cell wall structure. Additionally, we identified NAM recycling enzyme homologues in T. forsythia that are similar to the enzymes found in Pseudomonas putida. These homologues were then genetically transformed into a laboratory safe Escherichia coli strain, resulting in the efficient incorporation of unnatural NAM analogues into the peptidoglycan backbone and its visualization, alone or in the presence of human macrophages. This strain will be useful in further studies to probe NAM recycling and peptidoglycan scavenging pathways of T. forsythia and other cohabiting bacteria.

PMID:35924866 | PMC:PMC9464701 | DOI:10.1021/acsinfecdis.2c00333

Nat Rev Microbiol. 2022 Jul 25. doi: 10.1038/s41579-022-00763-4. Online ahead of print.

ABSTRACT

Mycobacterium tuberculosis, the causative agent of tuberculosis, has infected humans for millennia. M. tuberculosis is well adapted to establish infection, persist in the face of the host immune response and be transmitted to uninfected individuals. Its ability to complete this infection cycle depends on it both evading and taking advantage of host immune responses. The outcome of M. tuberculosis infection is often a state of equilibrium characterized by immunological control and bacterial persistence. Recent data have highlighted the diverse cell populations that respond to M. tuberculosis infection and the dynamic changes in the cellular and intracellular niches of M. tuberculosis during the course of infection. M. tuberculosis possesses an arsenal of protein and lipid effectors that influence macrophage functions and inflammatory responses; however, our understanding of the role that specific bacterial virulence factors play in the context of diverse cellular reservoirs and distinct infection stages is limited. In this Review, we discuss immune evasion and provocation by M. tuberculosis during its infection cycle and describe how a more detailed molecular understanding is crucial to enable the development of novel host-directed therapies, disease biomarkers and effective vaccines.

PMID:35879556 | DOI:10.1038/s41579-022-00763-4

Nat Biotechnol. 2022 Jul 25. doi: 10.1038/s41587-022-01384-1. Online ahead of print.

ABSTRACT

The use of therapeutic monoclonal antibodies is constrained because single antigen targets often do not provide sufficient selectivity to distinguish diseased from healthy tissues. We present HexElect^®, an approach to enhance the functional selectivity of therapeutic antibodies by making their activity dependent on clustering after binding to two different antigens expressed on the same target cell. lmmunoglobulin G (lgG)-mediated clustering of membrane receptors naturally occurs on cell surfaces to trigger complement- or cell-mediated effector functions or to initiate intracellular signaling. We engineer the Fc domains of two different lgG antibodies to suppress their individual homo-oligomerization while promoting their pairwise hetero-oligomerization after binding co-expressed antigens. We show that recruitment of complement component C1q to these hetero-oligomers leads to clustering-dependent activation of effector functions such as complement mediated killing of target cells or activation of cell surface receptors. HexElect allows selective antibody activity on target cells expressing unique, potentially unexplored combinations of surface antigens.

PMID:35879362 | DOI:10.1038/s41587-022-01384-1

ACS Omega. 2022 Jul 11;7(28):24111-24120. doi: 10.1021/acsomega.1c07360. eCollection 2022 Jul 19.

ABSTRACT

Surface-expressed bacterial polysaccharides are important vaccine antigens but must be conjugated to a carrier protein for efficient antigen presentation and development of strong memory B cell and antibody responses, especially in young children. The commonly used protein carriers include tetanus toxoid (TT), diphtheria toxoid (DT), and its derivative CRM197, but carrier-induced epitopic suppression and bystander interference may limit the expanded use of the same carriers in the pediatric immunization schedule. Recent efforts to develop a vaccine against the major human pathogen group A Streptococcus (GAS) have sought to combine two promising vaccine antigens-the universally conserved group A cell wall carbohydrate (GAC) with the secreted toxin antigen streptolysin O (SLO) as a protein carrier; however, standard reductive amination procedures appeared to destroy function epitopes of the protein, markedly diminishing functional antibody responses. Here, we couple a cell-free protein synthesis (CFPS) platform, allowing the incorporation of non-natural amino acids into a C-terminally truncated SLO toxoid for the precise conjugation to the polyrhamnose backbone of GAC. The combined immunogen generated functional antibodies against both conserved GAS virulence factors and provided protection against systemic GAS challenges. CFPS may represent a scalable method for generating pathogen-specific carrier proteins for multivalent subunit vaccine development.

PMID:35874267 | PMC:PMC9301713 | DOI:10.1021/acsomega.1c07360