Animal models of shigellosis: a historical overview.

Shigella spp. are major causative agents of bacillary dysentery, a severe enteric disease characterized by destruction and inflammation of the colonic epithelium accompanied by acute diarrhea, fever, and abdominal pain. Although antibiotics have traditionally been effective, the prevalence of multidrug-resistant strains is increasing, stressing the urgent need for a vaccine. The human-specific nature of shigellosis and the absence of a dependable animal model have posed significant obstacles in understanding Shigella pathogenesis and the host immune response, both of which are crucial for the development of an effective vaccine. Efforts have been made over time to develop a physiological model that mimics the pathological features of the human disease with limited success until the recent development of genetically modified mouse models. In this review, we provide an overview of Shigella pathogenesis and chronicle the historical development of various shigellosis models, emphasizing their strengths and weaknesses.

A Nuclear Export Signal in KHNYN Required for Its Antiviral Activity Evolved as ZAP Emerged in Tetrapods.

The zinc finger antiviral protein (ZAP) inhibits viral replication by directly binding CpG dinucleotides in cytoplasmic viral RNA to inhibit protein synthesis and target the RNA for degradation. ZAP evolved in tetrapods and there are clear orthologs in reptiles, birds, and mammals. When ZAP emerged, other proteins may have evolved to become cofactors for its antiviral activity. KHNYN is a putative endoribonuclease that is required for ZAP to restrict retroviruses. To determine its evolutionary path after ZAP emerged, we compared KHNYN orthologs in mammals and reptiles to those in fish, which do not encode ZAP. This identified residues in KHNYN that are highly conserved in species that encode ZAP, including several in the CUBAN domain. The CUBAN domain interacts with NEDD8 and Cullin-RING E3 ubiquitin ligases. Deletion of the CUBAN domain decreased KHNYN antiviral activity, increased protein expression and increased nuclear localization. However, mutation of residues required for the CUBAN domain-NEDD8 interaction increased KHNYN abundance but did not affect its antiviral activity or cytoplasmic localization, indicating that Cullin-mediated degradation may control its homeostasis and regulation of protein turnover is separable from its antiviral activity. By contrast, the C-terminal residues in the CUBAN domain form a CRM1-dependent nuclear export signal (NES) that is required for its antiviral activity. Deletion or mutation of the NES increased KHNYN nuclear localization and decreased its interaction with ZAP. The final 2 positions of this NES are not present in fish KHNYN orthologs and we hypothesize their evolution allowed KHNYN to act as a ZAP cofactor. IMPORTANCE The interferon system is part of the innate immune response that inhibits viruses and other pathogens. This system emerged approximately 500 million years ago in early vertebrates. Since then, some genes have evolved to become antiviral interferon-stimulated genes (ISGs) while others evolved so their encoded protein could interact with proteins encoded by ISGs and contribute to their activity. However, this remains poorly characterized. ZAP is an ISG that arose during tetrapod evolution and inhibits viral replication. Because KHNYN interacts with ZAP and is required for its antiviral activity against retroviruses, we conducted an evolutionary analysis to determine how specific amino acids in KHNYN evolved after ZAP emerged. This identified a nuclear export signal that evolved in tetrapods and is required for KHNYN to traffic in the cell and interact with ZAP. Overall, specific residues in KHNYN evolved to allow it to act as a cofactor for ZAP antiviral activity.

Ca2+-calmodulin signalling at the host-pathogen interface.

Multiple eukaryotic cell processes are modulated by calcium ions (Ca2+). As such, Ca2+ is emerging as a crucial regulator of innate immunity in multicellular organisms. In particular, recent studies have identified roles of Ca2+ signalling at the host-bacteria interface. Following microbial exposure, Ca2+ signals mobilised from the extracellular milieu or intracellular stores are transduced into cell physiological responses. However, during infection with host-adapted pathogens, Ca2+ signals are often atypical, due to the activities of virulence factors, with varied consequences for both the pathogen and the host cell. In this review, we describe the Ca2+-dependent host factors regulating antibacterial immunity, in addition to bacterial effectors that promote, inhibit, or co-opt Ca2+-calmodulin signalling to promote infection.

Meeting report - Cell dynamics: host-pathogen interface.

Two years into the most significant infectious disease event of our generation, infections have populated every conversation and in-depth understanding of host-pathogen interactions has, perhaps, never been more important. In a successful return to in-person conferences, the host-pathogen interface was the focus of the third Cell Dynamics meeting, which took place at the glorious Wotton House in Surrey, UK. The meeting organised by Michaela Gack, Maximiliano Gutierrez, Dominique Soldati-Favre and Michael Way gathered an international group of scientists who shared their recent discoveries and views on numerous aspects, including cell-autonomous defence mechanisms, pathogen interactions with host cytoskeletal or membrane dynamics, and cellular immune regulation. More than 30 years into the beginning of cellular microbiology as a field, the meeting exhibited the unique aspect of the host-pathogen interface in uncovering the fundamentals of both pathogens and their hosts.

Functions of IFNλs in Anti-Bacterial Immunity at Mucosal Barriers.

Type III interferons (IFNs), or IFNλs, are cytokines produced in response to microbial ligands. They signal through the IFNλ receptor complex (IFNLR), which is located on epithelial cells and select immune cells at barrier sites. As well as being induced during bacterial or viral infection, type III IFNs are produced in response to the microbiota in the lung and intestinal epithelium where they cultivate a resting antiviral state. While the multiple anti-viral activities of IFNλs have been extensively studied, their roles in immunity against bacteria are only recently emerging. Type III IFNs increase epithelial barrier integrity and protect from infection in the intestine but were shown to increase susceptibility to bacterial superinfections in the respiratory tract. Therefore, the effects of IFNλ can be beneficial or detrimental to the host during bacterial infections, depending on timing and biological contexts. This duality will affect the potential benefits of IFNλs as therapeutic agents. In this review, we summarize the current knowledge on IFNλ induction and signaling, as well as their roles at different barrier sites in the context of anti-bacterial immunity.

A family of conserved bacterial virulence factors dampens interferon responses by blocking calcium signaling.

Interferons (IFNs) induce an antimicrobial state, protecting tissues from infection. Many viruses inhibit IFN signaling, but whether bacterial pathogens evade IFN responses remains unclear. Here, we demonstrate that the Shigella OspC family of type-III-secreted effectors blocks IFN signaling independently of its cell death inhibitory activity. Rather, IFN inhibition was mediated by the binding of OspC1 and OspC3 to the Ca2+ sensor calmodulin (CaM), blocking CaM kinase II and downstream JAK/STAT signaling. The growth of Shigella lacking OspC1 and OspC3 was attenuated in epithelial cells and in a murine model of infection. This phenotype was rescued in both models by the depletion of IFN receptors. OspC homologs conserved in additional pathogens not only bound CaM but also inhibited IFN, suggesting a widespread virulence strategy. These findings reveal a conserved but previously undescribed molecular mechanism of IFN inhibition and demonstrate the critical role of Ca2+ and IFN targeting in bacterial pathogenesis.

Reprogramming of Cell Death Pathways by Bacterial Effectors as a Widespread Virulence Strategy.

The modulation of programmed cell death (PCD) processes during bacterial infections is an evolving arms race between pathogens and their hosts. The initiation of apoptosis, necroptosis, and pyroptosis pathways are essential to immunity against many intracellular and extracellular bacteria. These cellular self-destructive mechanisms are used by the infected host to restrict and eliminate bacterial pathogens. Without a tight regulatory control, host cell death can become a double-edged sword. Inflammatory PCDs contribute to an effective immune response against pathogens, but unregulated inflammation aggravates the damage caused by bacterial infections. Thus, fine-tuning of these pathways is required to resolve infection while preserving the host immune homeostasis. In turn, bacterial pathogens have evolved secreted virulence factors or effector proteins that manipulate PCD pathways to promote infection. In this review, we discuss the importance of controlled cell death in immunity to bacterial infection. We also detail the mechanisms employed by type 3 secreted bacterial effectors to bypass these pathways and their importance in bacterial pathogenesis.

Interferons: Tug of War Between Bacteria and Their Host.

Type I and III interferons (IFNs) are archetypally antiviral cytokines that are induced in response to recognition of foreign material by pattern recognition receptors (PRRs). Though their roles in anti-viral immunity are well established, recent evidence suggests that they are also crucial mediators of inflammatory processes during bacterial infections. Type I and III IFNs restrict bacterial infection in vitro and in some in vivo contexts. IFNs mainly function through the induction of hundreds of IFN-stimulated genes (ISGs). These include PRRs and regulators of antimicrobial signaling pathways. Other ISGs directly restrict bacterial invasion or multiplication within host cells. As they regulate a diverse range of anti-bacterial host responses, IFNs are an attractive virulence target for bacterial pathogens. This review will discuss the current understanding of the bacterial effectors that manipulate the different stages of the host IFN response: IFN induction, downstream signaling pathways, and target ISGs.

COVID-19 and emerging viral infections: The case for interferon lambda.

With the first reports on coronavirus disease 2019 (COVID-19), which is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the scientific community working in the field of type III IFNs (IFN-λ) realized that this class of IFNs could play an important role in this and other emerging viral infections. In this Viewpoint, we present our opinion on the benefits and potential limitations of using IFN-λ to prevent, limit, and treat these dangerous viral infections.

Asymmetric distribution of TLR3 leads to a polarized immune response in human intestinal epithelial cells.

Intestinal epithelial cells (IECs) act as a physical barrier separating the commensal-containing intestinal tract from the sterile interior. These cells have found a complex balance allowing them to be prepared for pathogen attacks while still tolerating the presence of bacterial or viral stimuli present in the lumen of the gut. Using primary human IECs, we probed the mechanisms that allow for such a tolerance. We discovered that viral infections emanating from the basolateral side of IECs elicit a stronger intrinsic immune response in comparison to lumenal apical infections. We determined that this asymmetric immune response is driven by the clathrin-sorting adaptor AP-1B, which mediates the polarized sorting of Toll-like receptor 3 (TLR3) towards the basolateral side of IECs. Mice and human IECs lacking AP-1B showed an exacerbated immune response following apical stimulation. Together, these results suggest a model where the cellular polarity program plays an integral role in the ability of IECs to partially tolerate apical commensals while remaining fully responsive to invasive basolateral pathogens.

Host-Encoded Sensors of Bacteria: Our Windows into the Microbial World.

Bacterial pathogens can be very efficient at causing disease and are the cause of some of the worst epidemics that have affected humanity. However, most infections are prevented by the actions of our immune system. Immune activation depends on the rapid detection of bacteria by a diverse family of sensory proteins known as pattern recognition receptors. These receptors detect conserved features of bacteria that are not found in humans but are often necessary for survival within the host or environment. In this review, we discuss the strategies used by pattern recognition receptors to detect bacteria and their products. We also discuss emerging evidence that some pattern recognition receptors can be activated by bacterial pathogens specifically, through the surveillance of host activities that are commonly targeted by virulence factors. This collection of surveillance mechanisms provides an interconnected network of defense, which is important to maintain the germ-free environment of the inner organs of humans and other multicellular organisms.

Increasing the CpG dinucleotide abundance in the HIV-1 genomic RNA inhibits viral replication.

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) structural protein Gag is necessary and sufficient to form viral particles. In addition to encoding the amino acid sequence for Gag, the underlying RNA sequence could encode cis-acting elements or nucleotide biases that are necessary for viral replication. Furthermore, RNA sequences that inhibit viral replication could be suppressed in gag. However, the functional relevance of RNA elements and nucleotide biases that promote or repress HIV-1 replication remain poorly understood. RESULTS: To characterize if the RNA sequence in gag controls HIV-1 replication, the matrix (MA) region was codon modified, allowing the RNA sequence to be altered without affecting the protein sequence. Codon modification of nucleotides (nt) 22-261 or 22-378 in gag inhibited viral replication by decreasing genomic RNA (gRNA) abundance, gRNA stability, Gag expression, virion production and infectivity. Comparing the effect of these point mutations to deletions of the same region revealed that the mutations inhibited infectious virus production while the deletions did not. This demonstrated that codon modification introduced inhibitory sequences. There is a much lower than expected frequency of CpG dinucleotides in HIV-1 and codon modification introduced a substantial increase in CpG abundance. To determine if they are necessary for inhibition of HIV-1 replication, codons introducing CpG dinucleotides were mutated back to the wild type codon, which restored efficient Gag expression and infectious virion production. To determine if they are sufficient to inhibit viral replication, CpG dinucleotides were inserted into gag in the absence of other changes. The increased CpG dinucleotide content decreased HIV-1 infectivity and viral replication. CONCLUSIONS: The HIV-1 RNA sequence contains low abundance of CpG dinucleotides. Increasing the abundance of CpG dinucleotides inhibits multiple steps of the viral life cycle, providing a functional explanation for why CpG dinucleotides are suppressed in HIV-1.

Type III IFNs Are Commonly Induced by Bacteria-Sensing TLRs and Reinforce Epithelial Barriers during Infection.

Type III IFNs (IFN-λs) are secreted factors that are well-known for their antiviral activities. However, their regulation and functions during bacterial infections are unclear. In this article, we report that the regulation of IFN-λ genes did not track with mechanisms that control type I IFN expression in response to TLRs. Whereas type I IFNs were only expressed from TLRs present on endosomes, type III IFNs could be induced by TLRs that reside at the plasma membrane and that detect various bacterial products. The mechanisms that regulate type III IFN gene expression tracked with those that promote inflammatory cytokine and chemokine expression. Importantly, rIFN-λs enhanced epithelial barriers in vitro, preventing transcellular bacteria dissemination. We therefore propose that in addition to their functions in cell-intrinsic antiviral immunity, type III IFNs protect epithelial barrier integrity, an activity that would benefit the host during any infectious encounter.

Activation and pathogenic manipulation of the sensors of the innate immune system.

The innate immune system detects the presence of microbes through different families of pattern-recognition receptors (PRRs). PRRs detect pathogens of all origins and trigger signaling events that activate innate and adaptive immunity. These events need to be tightly regulated in order to ensure optimal activation when required, and minimal signaling in the absence of microbial encounters. This regulation is achieved, at least in part, through the precise subcellular positioning of receptors and downstream signaling proteins. Consequently, mislocalization of these proteins inhibits innate immune pathways, and pathogens have evolved to alter host protein localization as a strategy to evade immune detection. This review describes the importance of subcellular localization of various PRR families and their adaptors, and highlights pathogenic immune evasion strategies that operate by altering immune protein localization.

A Single Bacterial Immune Evasion Strategy Dismantles Both MyD88 and TRIF Signaling Pathways Downstream of TLR4.

During bacterial infections, Toll-like receptor 4 (TLR4) signals through the MyD88- and TRIF-dependent pathways to promote pro-inflammatory and interferon (IFN) responses, respectively. Bacteria can inhibit the MyD88 pathway, but if the TRIF pathway is also targeted is unclear. We demonstrate that, in addition to MyD88, Yersinia pseudotuberculosis inhibits TRIF signaling through the type III secretion system effector YopJ. Suppression of TRIF signaling occurs during dendritic cell (DC) and macrophage infection and prevents expression of type I IFN and pro-inflammatory cytokines. YopJ-mediated inhibition of TRIF prevents DCs from inducing natural killer (NK) cell production of antibacterial IFNγ. During infection of DCs, YopJ potently inhibits MAPK pathways but does not prevent activation of IKK- or TBK1-dependent pathways. This singular YopJ activity efficiently inhibits TLR4 transcription-inducing activities, thus illustrating a simple means by which pathogens impede innate immunity.

The unique regulation and functions of type III interferons in antiviral immunity.

Type I interferons (IFNs) were long considered to be the sole IFN species produced by virus-infected cells until the discovery of type III IFNs (IFNλs), decades later. Like type I IFNs, type III IFNs are induced by and protect against viral infections, leading to the initial conclusion that the two IFN species are identical in regulation and biological functions. However, the two systems differ in the tissue expression of their receptor, resulting in different roles in vivo. The unique nature of IFNλs has been further demonstrated by recent studies revealing differences in the regulation of type I and III IFN expression, and how these proteins elicit specific cellular responses. This review focuses on the distinctive features of type III IFNs in antiviral innate immunity.

Diverse intracellular pathogens activate type III interferon expression from peroxisomes.

Type I interferon responses are considered the primary means by which viral infections are controlled in mammals. Despite this view, several pathogens activate antiviral responses in the absence of type I interferons. The mechanisms controlling type I interferon-independent responses are undefined. We found that RIG-I like receptors (RLRs) induce type III interferon expression in a variety of human cell types, and identified factors that differentially regulate expression of type I and type III interferons. We identified peroxisomes as a primary site of initiation of type III interferon expression, and revealed that the process of intestinal epithelial cell differentiation upregulates peroxisome biogenesis and promotes robust type III interferon responses in human cells. These findings highlight the importance of different intracellular organelles in specific innate immune responses.

Peroxisomes and the antiviral responses of mammalian cells.

Cell biology and microbiology are some of the oldest areas of scientific inquiry. Despite the depth of knowledge we now have in these respective fields, much remains unclear about how microorganisms interact with host intracellular organelles. Perhaps nowhere is this statement more accurate than in the role of peroxisomes in microbial infections. Peroxisomes were one of the first organelles discovered by Christian De Duve over 50 years ago (de Duve Ann N Y Acad Sci 386:1-4, 1982). These organelles are ubiquitously found in eukaryotic cells, where they serve several well-defined functions in lipid and oxygen homeostasis (Waterham and Wanders Biochim Biophys Acta 1822:1325, 2012). This chapter will discuss the emerging evidence that indicates that in addition to their functions in cellular metabolism, peroxisomes play an important role in viral infections.