Molecular mechanism of Streptococcus pneumoniae-targeting xenophagy recognition and evasion: Reinterpretation of pneumococci as intracellular bacteria.

Streptococcus pneumoniae is a major, encapsulated Gram-positive pathogen that causes diseases including community-acquired pneumonia, meningitis, and sepsis. This pathogen colonizes the nasopharyngeal epithelia asymptomatically but can often migrate to sterile tissues and cause life-threatening invasive infections (invasive pneumococcal disease). Although multivalent pneumococcal polysaccharides and conjugate vaccines are available and effective, they also have major shortcomings with respect to the emergence of vaccine-resistant serotypes. Therefore, alternative therapeutic approaches are needed, and the molecular analysis of host-pathogen interactions and their applications to pharmaceutical development and clinical practice has recently received increased attention. In this review, we introduce pneumococcal surface virulence factors involved in pathogenicity and highlight recent advances in our understanding of host autophagy recognition mechanisms against intracellular S. pneumoniae and pneumococcal evasion from autophagy.

Invasive Streptococcus oralis Expressing Serotype 3 Pneumococcal Capsule, Japan.

We report 2 adult cases of invasive disease in Japan caused by Streptococcus oralis that expressed the serotype 3 pneumococcal capsule and formed mucoid colonies. Whole-genome sequencing revealed that the identical serotype 3 pneumococcal capsule locus and hyl fragment were recombined into the genomes of 2 distinct S. oralis strains.

Streptococcus pneumoniae hijacks host autophagy by deploying CbpC as a decoy for Atg14 depletion.

Pneumococcal cell surface-exposed choline-binding proteins (CBPs) play pivotal roles in multiple infectious processes with pneumococci. Intracellular pneumococci can be recognized at multiple steps during bactericidal autophagy. However, whether CBPs are involved in pneumococci-induced autophagic processes remains unknown. In this study, we demonstrate that CbpC from S. pneumoniae strain TIGR4 activates autophagy through an interaction with Atg14. However, S. pneumoniae also interferes with autophagy by deploying CbpC as a decoy to cause autophagic degradation of Atg14 through an interaction with p62/SQSTM1. Thus, S. pneumoniae suppresses the autophagic degradation of intracellular pneumococci and survives within cells. Domain analysis reveals that the coiled-coil domain of Atg14 and residue Y83 of the dp3 domain in the N-terminal region of CbpC are crucial for both the CbpC-Atg14 interaction and the subsequent autophagic degradation of Atg14. Although homology modeling indicates that CbpC orthologs have similar structures in the dp3 domain, autophagy induction through Atg14 binding is an intrinsic property of CbpC. Our data provide novel insights into the evolutionary hijacking of host-defense systems by intracellular pneumococci.

Molecular mechanisms of Streptococcus pneumoniae-targeted autophagy via pneumolysin, Golgi-resident Rab41, and Nedd4-1-mediated K63-linked ubiquitination.

Streptococcus pneumoniae is the most common causative agent of community-acquired pneumonia and can penetrate epithelial barriers to enter the bloodstream and brain. We investigated intracellular fates of S. pneumoniae and found that the pathogen is entrapped by selective autophagy in pneumolysin- and ubiquitin-p62-LC3 cargo-dependent manners. Importantly, following induction of autophagy, Rab41 was relocated from the Golgi apparatus to S. pneumoniae-containing autophagic vesicles (PcAV), which were only formed in the presence of Rab41-positive intact Golgi apparatuses. Moreover, subsequent localization and regulation of K48- and K63-linked polyubiquitin chains in and on PcAV were clearly distinguishable from each other. Finally, we found that E3 ligase Nedd4-1 was recruited to PcAV and played a pivotal role in K63-linked polyubiquitin chain (K63Ub) generation on PcAV, promotion of PcAV formation, and elimination of intracellular S. pneumoniae. These findings suggest that Nedd4-1-mediated K63Ub deposition on PcAV acts as a scaffold for PcAV biogenesis and efficient elimination of host cell-invaded pneumococci.

The Shigella flexneri effector OspI deamidates UBC13 to dampen the inflammatory response.

Many bacterial pathogens can enter various host cells and then survive intracellularly, transiently evade humoral immunity, and further disseminate to other cells and tissues. When bacteria enter host cells and replicate intracellularly, the host cells sense the invading bacteria as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) by way of various pattern recognition receptors. As a result, the host cells induce alarm signals that activate the innate immune system. Therefore, bacteria must modulate host inflammatory signalling and dampen these alarm signals. How pathogens do this after invading epithelial cells remains unclear, however. Here we show that OspI, a Shigella flexneri effector encoded by ORF169b on the large plasmid and delivered by the type ΙΙΙ secretion system, dampens acute inflammatory responses during bacterial invasion by suppressing the tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6)-mediated signalling pathway. OspI is a glutamine deamidase that selectively deamidates the glutamine residue at position 100 in UBC13 to a glutamic acid residue. Consequently, the E2 ubiquitin-conjugating activity required for TRAF6 activation is inhibited, allowing S. flexneri OspI to modulate the diacylglycerol-CBM (CARD-BCL10-MALT1) complex-TRAF6-nuclear-factor-κB signalling pathway. We determined the 2.0 Å crystal structure of OspI, which contains a putative cysteine-histidine-aspartic acid catalytic triad. A mutational analysis showed this catalytic triad to be essential for the deamidation of UBC13. Our results suggest that S. flexneri inhibits acute inflammatory responses in the initial stage of infection by targeting the UBC13-TRAF6 complex.

Shigella IpaH7.8 E3 ubiquitin ligase targets glomulin and activates inflammasomes to demolish macrophages.

When nucleotide-binding oligomerization domain-like receptors (NLRs) sense cytosolic-invading bacteria, they induce the formation of inflammasomes and initiate an innate immune response. In quiescent cells, inflammasome activity is tightly regulated to prevent excess inflammation and cell death. Many bacterial pathogens provoke inflammasome activity and induce inflammatory responses, including cell death, by delivering type III secreted effectors, the rod component flagellin, and toxins. Recent studies indicated that Shigella deploy multiple mechanisms to stimulate NLR inflammasomes through type III secretion during infection. Here, we show that Shigella induces rapid macrophage cell death by delivering the invasion plasmid antigen H7.8 (IpaH7.8) enzyme 3 (E3) ubiquitin ligase effector via the type III secretion system, thereby activating the NLR family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing 4 (NLRC4) inflammasomes and caspase-1 and leading to macrophage cell death in an IpaH7.8 E3 ligase-dependent manner. Mice infected with Shigella possessing IpaH7.8, but not with Shigella possessing an IpaH7.8 E3 ligase-null mutant, exhibited enhanced bacterial multiplication. We defined glomulin/flagellar-associated protein 68 (GLMN) as an IpaH7.8 target involved in IpaH7.8 E3 ligase-dependent inflammasome activation. This protein originally was identified through its association with glomuvenous malformations and more recently was described as a member of a Cullin ring ligase inhibitor. Modifying GLMN levels through overexpression or knockdown led to reduced or augmented inflammasome activation, respectively. Macrophages stimulated with lipopolysaccharide/ATP induced GLMN puncta that localized with the active form of caspase-1. Macrophages from GLMN(+/-) mice were more responsive to inflammasome activation than those from GLMN(+/+) mice. Together, these results highlight a unique bacterial adaptation that hijacks inflammasome activation via interactions between IpaH7.8 and GLMN.

Bacteria hijack integrin-linked kinase to stabilize focal adhesions and block cell detachment.

The rapid turnover and exfoliation of mucosal epithelial cells provides an innate defence system against bacterial infection. Nevertheless, many pathogenic bacteria, including Shigella, are able to surmount exfoliation and colonize the epithelium efficiently. Here we show that the Shigella flexneri effector OspE (consisting of OspE1 and OspE2 proteins), which is highly conserved among enteropathogenic Escherichia coli, enterohaemorrhagic E. coli, Citrobacter rodentium and Salmonella strains, reinforces host cell adherence to the basement membrane by interacting with integrin-linked kinase (ILK). The number of focal adhesions was augmented along with membrane fraction ILK by ILK-OspE binding. The interaction between ILK and OspE increased cell surface levels of 1 integrin and suppressed phosphorylation of focal adhesion kinase and paxillin, which are required for rapid turnover of focal adhesion in cell motility. Nocodazole-washout-induced focal adhesion disassembly was blocked by expression of OspE. Polarized epithelial cells infected with a Shigella mutant lacking the ospE gene underwent more rapid cell detachment than cells infected with wild-type Shigella. Infection of guinea pig colons with Shigella corroborated the pivotal role of the OspE-ILK interaction in suppressing epithelial detachment, increasing bacterial cell-to-cell spreading, and promoting bacterial colonization. These results indicate that Shigella sustain their infectious foothold by using special tactics to prevent detachment of infected cells.

Evaluation of streptococcal toxic shock-like syndrome caused by group B streptococcus in adults in Japan between 2009 and 2013.

Infection with Streptococcus agalactiae has long been recognized in infants. In recent years, S. agalactiae is an important cause of morbidity and mortality among adults and among those with underlying medical condition. Several cases of GBS infection and more fulminant disease similar to streptococcal toxic shock syndrome have recently been reported. We report here that 19 S. agalactiae strains were isolated from streptococcal toxic shock-like syndrome cases involving adult patients in Japan between 2009 and 2013. The average age of the patients was 66.3 years. At least one underlying disease was present in 47.4% (9/19) of the patients. The most prevalent serotype among these strains was Ib. All serotype Ib strains belonged to clonal complex 10 and were ciprofloxacin resistant. In contrast, all strains were susceptible to penicillin G, ampicillin, cefazolin, cefotaxime, imipenem, panipenem, and linezolid. The characteristic type distributions of streptococcal toxic shock-like syndrome isolates differed between isolates obtained from vaginal swabs of women and infants with invasive infections.

Individual Atg8 paralogs exhibit unique properties in streptococcus pneumoniae-induced hierarchical autophagy.

Individual Atg8 (autophagy related 8) paralogs, comprising MAP1LC3A/LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2/GATE16, play a crucial role in canonical macroautophagy/autophagy. However, their functions remain unclear owing to functional redundancy. In a previous study, we reported that intracellular Streptococcus pneumoniae triggers hierarchical autophagy in response to bacterial infection. This process commences with the induction of conjugation of Atg8 paralogs (Atg8s) to single membranes (CASM), followed by CASM shedding and subsequent induction of xenophagy. In our recent study, we performed functional analysis of Atg8s during pneumococci-induced hierarchical autophagy. Our findings suggest that LC3A and GABARAPL1 are crucial for CASM induction, whereas GABARAPL2 and GABARAP play sequential roles in CASM shedding and subsequent induction of xenophagy, respectively.Abbreviation: Atg8: autophagy related 8; Atg8s: Atg8 paralogs; CASM: conjugation of Atg8s to single membranes; mpi: minutes post-infection; mpi: minutes post-infection; PcAV: pneumococci-containing autophagic vesicles; PcLV: LC3-associated phagosome (LAPosome)-like vacuole; PcV: pneumococci-containing vesicles; Sp: S. pneumoniae.

Clinical features relating to pneumococcal colony phase variation in hospitalized adults with pneumonia.

Background. Streptococcus pneumoniae is a major causative bacteria of pneumonia and invasive pneumococcal disease (IPD); however, the mechanisms underlying its severity and invasion remain to be defined. Pneumococcal colonies exhibit opaque and transparent opacity phase variations, which have been associated with invasive infections and nasal colonization, respectively, in animal studies. This study evaluated the relationship between the opacity of pneumococcal colonies and the clinical presentation of pneumococcal pneumonia.Methods. This retrospective study included adult patients hospitalized with pneumococcal pneumonia between 2012 and 2019 at four tertiary medical institutions. Pneumococcal strains from lower respiratory tract specimens were determined for their serotypes and microscopic colony opacity, and the association between the opacity phase and the severity of pneumonia was evaluated. Serotypes 3 and 37 with mucoid colony phenotypes were excluded from the study because their colony morphologies were clearly different.Results. A total of 92 patients were included. Most patients were older adults (median age: 72 years) and males (67 %), and 59 % had community-acquired pneumonia. Of the 92 patients, 41 (45 %), 12 (13 %), and 39 (42 %) patients had opaque, transparent, and mixed variants in their pneumococcal colony, respectively. The opaque and non-opaque pneumococcal variants had no statistically significant difference in patient backgrounds. Although the pneumonia severity index score did not differ between the opaque and non-opaque groups, the rate of bacteremia was significantly higher in the opaque group than in the non-opaque group. Serotype distribution was similar between the groups.Conclusions. Opaque pneumococcal variants may cause pneumonia and invasive diseases in humans. This study could help elucidate IPD, and opacity assessment may serve as a predictor for IPD.

Individual Atg8 paralogs and a bacterial metabolite sequentially promote hierarchical CASM-xenophagy induction and transition.

Atg8 paralogs, consisting of LC3A/B/C and GBRP/GBRPL1/GATE16, function in canonical autophagy; however, their function is controversial because of functional redundancy. In innate immunity, xenophagy and non-canonical single membranous autophagy called "conjugation of Atg8s to single membranes" (CASM) eliminate bacteria in various cells. Previously, we reported that intracellular Streptococcus pneumoniae can induce unique hierarchical autophagy comprised of CASM induction, shedding, and subsequent xenophagy. However, the molecular mechanisms underlying these processes and the biological significance of transient CASM induction remain unknown. Herein, we profile the relationship between Atg8s, autophagy receptors, poly-ubiquitin, and Atg4 paralogs during pneumococcal infection to understand the driving principles of hierarchical autophagy and find that GATE16 and GBRP sequentially play a pivotal role in CASM shedding and subsequent xenophagy induction, respectively, and LC3A and GBRPL1 are involved in CASM/xenophagy induction. Moreover, we reveal ingenious bacterial tactics to gain intracellular survival niches by manipulating CASM-xenophagy progression by generating intracellular pneumococci-derived H2O2.

Pneumococcal sialidase promotes bacterial survival by fine-tuning of pneumolysin-mediated membrane disruption.

Pneumolysin (Ply) is an indispensable cholesterol-dependent cytolysin for pneumococcal infection. Although Ply-induced disruption of pneumococci-containing endosomal vesicles is a prerequisite for the evasion of endolysosomal bacterial clearance, its potent activity can be a double-edged sword, having a detrimental effect on bacterial survivability by inducing severe endosomal disruption, bactericidal autophagy, and scaffold epithelial cell death. Thus, Ply activity must be maintained at optimal levels. We develop a highly sensitive assay to monitor endosomal disruption using NanoBiT-Nanobody, which shows that the pneumococcal sialidase NanA can fine-tune Ply activity by trimming sialic acid from cell-membrane-bound glycans. In addition, oseltamivir, an influenza A virus sialidase inhibitor, promotes Ply-induced endosomal disruption and cytotoxicity by inhibiting NanA activity in vitro and greater tissue damage and bacterial clearance in vivo. Our findings provide a foundation for innovative therapeutic strategies for severe pneumococcal infections by exploiting the duality of Ply activity.

Bacteria and host interactions in the gut epithelial barrier.

The gut mucosa acts as a barrier against microbial invaders, whereas resident commensal and foreign invading bacteria interact intimately with the gut epithelium and influence the host cellular and immune systems. The epithelial barrier serves as an infectious foothold for many bacterial pathogens and as an entry port for pathogens to disseminate into deeper tissues. Enteric bacterial pathogens can efficiently infect the gut mucosa using highly sophisticated virulence mechanisms that allow bacteria to circumvent the defense barriers in the gut. We provide an overview of the components of the mucosal barrier and discuss the bacterial stratagems that circumvent these barriers with particular emphasis on the roles of bacterial effector proteins.

Crosstalk between the innate immune system and selective autophagy in hepatitis B virus infection.

Although the involvement of macroautophagy/autophagy in hepatitis B virus (HBV) infection has become clearer recently, whether selective autophagy plays an important role in suppressing HBV remains uncertain. We recently found that LGALS9 (galectin 9) is an interferon (IFN)-inducible protein involved in the suppression of HBV replication. Expression of LGALS9 in HBV-infected cells causes the formation of cytoplasmic puncta that degrade the HBV core protein (HBc) in conjunction with RSAD2/viperin, another IFN-inducible protein. LGALS9 binds to HBc via RSAD2 and promotes the autoubiquitination of RNF13 (ring finger protein 13) to recruit SQSTM1/p62, resulting in the formation of LC3-positive autophagosomes that degrade HBc. Both LGALS9 and RSAD2 are encoded by IFN-stimulated genes that act synergistically to induce HBc proteolysis in HBV-infected hepatocytes in an IFN-dependent manner. These results reveal a crosstalk mechanism between the innate immune system and selective autophagy during viral infection.

Galectin-9 restricts hepatitis B virus replication via p62/SQSTM1-mediated selective autophagy of viral core proteins.

Autophagy has been linked to a wide range of functions, including a degradative process that defends host cells against pathogens. Although the involvement of autophagy in HBV infection has become apparent, it remains unknown whether selective autophagy plays a critical role in HBV restriction. Here, we report that a member of the galectin family, GAL9, directs the autophagic degradation of HBV HBc. BRET screening revealed that GAL9 interacts with HBc in living cells. Ectopic expression of GAL9 induces the formation of HBc-containing cytoplasmic puncta through interaction with another antiviral factor viperin, which co-localized with the autophagosome marker LC3. Mechanistically, GAL9 associates with HBc via viperin at the cytoplasmic puncta and enhanced the auto-ubiquitination of RNF13, resulting in p62 recruitment to form LC3-positive autophagosomes. Notably, both GAL9 and viperin are type I IFN-stimulated genes that act synergistically for the IFN-dependent proteolysis of HBc in HBV-infected hepatocytes. Collectively, these results reveal a previously undescribed antiviral mechanism against HBV in infected cells and a form of crosstalk between the innate immune system and selective autophagy in viral infection.

Manipulation of autophagy by bacteria for their own benefit.

Autophagy is the host innate immune system's first line of defense against microbial intruders. When the innate defense system recognizes invading bacterial pathogens and their infection processes, autophagic proteins act as cytosolic sensors that allow the autophagic pathway to be rapidly activated. However, many intracellular bacterial pathogens deploy highly evolved mechanisms to evade autophagic recognition, manipulate the autophagic pathway, and remodel the autophagosomal compartment for their own benefit. Here current topics regarding the recognition of invasive bacteria by the cytosolic innate immune system are highlighted, including autophagy and the mechanisms that enable bacteria to evade autophagy. Also highlighted are some selective examples of bacterial activities that manipulate the autophagic pathways for their own benefit.

Shigella infection of intestinal epithelium and circumvention of the host innate defense system.

Shigella, Gram-negative bacteria closely related to Escherichia coli, are highly adapted human pathogens that cause bacillary dysentery. Although Shigella have neither adherence factors nor flagella required for attaching or accessing the intestinal epithelium, Shigella are capable of colonizing the intestinal epithelium by exploiting epithelial-cell functions and circumventing the host innate immune response. During Shigella infection, they deliver many numbers of effectors through the type III secretion system into the surrounding space and directly into the host-cell cytoplasm. The effectors play pivotal roles from the onset of bacterial infection through to the establishment of the colonization of the intestinal epithelium, such as bacterial invasion, intracellular survival, subversion of the host immune defense response, and maintenance of the infectious foothold. These examples suggest that Shigella have evolved highly sophisticated infectious and intracellular strategies to establish replicative niches in the intestinal epithelium.

The multi-step mechanism and biological role of noncanonical autophagy targeting Streptococcus pneumoniae during the early stages of infection.

Multiple autophagic processes are triggered in response to bacterial infection as the host attempts to eliminate intracellular invaders. However, it is still unclear how the mechanisms contributing to canonical macroautophagy/autophagy, including xenophagy, coordinate with the more recently described features that are characteristic of noncanonical autophagy. Recently, we revealed that infection with Streptococcus pneumoniae can trigger the formation of RB1CC1/FIP200-independent LC3-associated phagosome-like vacuoles (PcLVs) that contain the pneumococci at an early stage of infection. We also found that interactions of SQSTM1/p62 with the ATG16L1 WD domain are essential for PcLV formation. Intriguingly, PcLVs were required for the subsequent generation of bactericidal autophagic vacuoles (PcAVs). Furthermore, we also identified LC3-delocalized SQSTM1-positive PcLVs as intracellular intermediates that link PcLVs and PcAVs. These findings reveal a novel multi-step mechanism that contributes to xenophagy of the critical S. pneumoniae respiratory pathogen.

Streptococcus pneumoniae promotes its own survival via choline-binding protein CbpC-mediated degradation of ATG14.

STREPTOCOCCUS PNEUMONIAE: is an opportunistic bacterial pathogen that can promote severe infection by overcoming the epithelial and blood-brain barrier. Pneumococcal cell-surface virulence factors, including cell wall-anchored choline-binding proteins (Cbps) play pivotal roles in promoting invasive disease. We reported previously that intracellular pneumococci were detected by hierarchical macroautophagic/autophagic processes that ultimately lead to bacterial elimination. However, whether intracellular pneumococci can evade autophagy by deploying Cbps remains unclear. In this study, we explore the biological functions of Cbps and reveal their roles in manipulating the autophagic process. Specifically, we found that CbpC-activated autophagy takes place via its interactions with ATG14 (autophagy related 14) and SQSTM1/p62 (sequestosome1). Importantly, CbpC dampens host autophagy by promoting ATG14 degradation via the ATG14-CbpC-SQSTM1/p62 axis. CbpC-induced reductions in ATG14 levels result in impaired ATG14-STX17 complex formation. In pneumococcal-infected cells, ATG14 levels are dramatically reduced in a CbpC-dependent manner that results in suppression of autophagy-mediated degradation and enhanced bacterial survival. Taken together, our results reveal a novel mechanism via which pneumococci can manipulate host autophagy responses, in this case, by employing CbpC as a trap to promote ATG14 depletion. Our findings highlight a novel and sophisticated tactic used by S. pneumoniae that serves to promote intracellular survival.

Streptococcus pneumoniae triggers hierarchical autophagy through reprogramming of LAPosome-like vesicles via NDP52-delocalization.

In innate immunity, multiple autophagic processes eliminate intracellular pathogens, but it remains unclear whether noncanonical autophagy and xenophagy are coordinated, and whether they occur concomitantly or sequentially. Here, we show that Streptococcus pneumoniae, a causative of invasive pneumococcal disease, can trigger FIP200-, PI3P-, and ROS-independent pneumococcus-containing LC3-associated phagosome (LAPosome)-like vacuoles (PcLVs) in an early stage of infection, and that PcLVs are indispensable for subsequent formation of bactericidal pneumococcus-containing autophagic vacuoles (PcAVs). Specifically, we identified LC3- and NDP52-delocalized PcLV, which are intermediates between PcLV and PcAV. Atg14L, Beclin1, and FIP200 were responsible for delocalizing LC3 and NDP52 from PcLVs. Thus, multiple noncanonical and canonical autophagic processes are deployed sequentially against intracellular S. pneumoniae. The Atg16L1 WD domain, p62, NDP52, and poly-Ub contributed to PcLV formation. These findings reveal a previously unidentified hierarchical autophagy mechanism during bactericidal xenophagy against intracellular bacterial pathogens, and should improve our ability to control life-threating pneumococcal diseases.