ELM provides a comprehensive, regularly updated and well-organised repository of manually curated, experimentally validated short linear motifs (SLiMs). An increasing number of SLiM-mediated interactions are discovered each year and keeping the resource up-to-date continues to be a great challenge. In the current update, 30 novel motif classes have been added and five existing classes have undergone major revisions. The update includes 411 new motif instances mostly focused on cell-cycle regulation, control of the actin cytoskeleton, membrane remodelling and vesicle trafficking pathways, liquid-liquid phase separation and integrin signalling. Many of the newly annotated motif-mediated interactions are targets of pathogenic motif mimicry by viral, bacterial or eukaryotic pathogens, providing invaluable insights into the molecular mechanisms underlying infectious diseases. The current ELM release includes 317 motif classes incorporating 3934 individual motif instances manually curated from 3867 scientific publications. ELM is available at:  Short linear motifs (SLiMs) are a distinct class of protein functional modules that participate in protein-protein interactions and act as sites of post-translational modification (PTM). The defining feature of SLiMs is their compact interfaces encoded in short linear stretches (commonly 3–15 residues long) of the protein sequence and the lack of a requirement for stable tertiary structure for their function, though many motifs fold upon binding. Most SLiMs are found within the structurally flexible and accessible intrinsically disordered regions (IDRs) of a proteome and are hallmarked by their evolutionary conservation in these rapidly evolving regions .SLiMs are core components across numerous cellular processes such as replication, differentiation and apoptosis. Their regulatory importance is highlighted by their role in vital cellular pathways, including cell cycle, endocytosis, cytoskeleton dynamics and intracellular signal transduction. The role of SLiMs in protein trafficking, post-translational modification and protein degradation underpins robust signalling regulation and contributes to spatiotemporal and contextual control of the signalling output . MutatioDiverse eukaryotic, bacterial and viral pathogens mimic SLiMs present in host cell proteins to hijack cellular signalling to their advantage as part of the infectious cycle ,7. The hConsidering the importance of SLiMs in cell regulation, the Eukaryotic Linear Motif (ELM) resource was created to scrutinise and systematically capture motif information from the literature. For nearly two decades, ELM has shared high-quality manually curated motif data with the community and has matured as the most widely used motif biology knowledgebase. Availability of these curated data provides avenues to discover novel SLiMs alongside serving as training data in developing bioinformatics toolkits and analysis workflows. In addition, the ELM web server allows users to search their proteins of interest to find candidate SLiMs that experimentalists can probe to test their role in cellular systems.In this paper, we report on the growth of the ELM dataset since our previous publication .https://en.wikipedia.org/wiki/Regular_expression and see the ‘Regular expressions’ section at http://elm.eu.org/infos/help.html for a detailed description of ELM consensus definitions) and derived from the evolutionary and structural information of the motif instances. ELM motif classes are further classified based on their high-level function into six broad categories: cleavage (CLV), degradation (DEG), docking (DOC), ligand (LIG), modification (MOD) and targeting (TRG) motifs.ELM (Eukaryotic Linear Motif) is a freely accessible resource for understanding and exploring the biology of SLiMs. At the core of the resource is a manually curated set of motif instances derived from the experimental literature ,13. Indihttp://www.postgresql.org/]. The data in the backend is accessed via the Django web framework [https://djangoproject.com/] and is served on the front end web interface designed for an intuitive and user-friendly browsing experience. The ELM resource also provides access to the functional site prediction toolkit, enabling users to search candidate motifs within their proteins of interest. Based on sequence matches to the motif regular expressions, the detected candidate motifs are mapped and shown onto the protein sequence. Furthermore, distinction between plausible true and false positive candidate motifs is made with logical filters based on globular domain, structure and contextual knowledge.Periodically, existing classes may be updated as new information becomes available. The resource stores the annotated data in a PostgreSQL relational database [http://elm.eu.org/downloads.html. In addition, a REST-API is available to programmatically search ELM class consensus matches against proteins of interest. The usage details of the motif search REST-API service are available at http://elm.eu.org/api/manual.html.The data stored in the ELM resource is freely accessible and downloadable in a range of formats. Details of the available formats and datasets can be found at Since the last release in 2020 , ELM curWhile drafting motif entries for ELM, several issues can make the task daunting and hamper the annotation process. One common problem is the lack of a clear identity for the protein being studied in the paper undergoing annotation. These issues are often related to the use of an ambiguous gene or protein name, or the omission of the source species. Other complicating scenarios appear when multiple potential matches for a motif or motif-binding domain are present in a protein and the paper's text does not clearly state which of the modules are being experimentally tested. In these cases it is sometimes not possible to annotate the motif instance or define the interacting partner domain region.Another commonly observed case deals with the mutagenesis of a predicted motif within the folded region of a protein. Such mutations will often partially or fully unfold a protein thereby altering protein function. As a result, the outcome from such a study will be misleading and will not help identify the actual functional motif region. In some cases the mutant phenotype can even match the expected phenotype for a motif mutation. For example, the mutagenesis of a predicted targeting motif buried in a folded region could result in protein unfolding and subsequent mislocalisation. The motif data is still extracted from such papers, and if they match the regular expression of the motif, they are assigned as false positives in ELM. In some articles, the experimental approaches applied do not conclusively prove the existence of a motif; for example, neither phenotypic or other complex functional assays nor simple peptide pull-downs from cell extracts unambiguously demonstrate the existence of SLiMs. These types of approach need to be complemented by more direct biophysical experiments.The ELM annotation workflow ensures quality assessment of the motifs by applying various measures. These involve assessing motifs for evolutionary conservation, surface accessibility, and inspecting structures for specificity determining residues, among other evaluations . AnnotatCell division is the universal process through which cells duplicate their genetic material and undergo cytokinesis to produce two daughter cells. While the details vary across eukaryotes, one major biochemical switch, the cyclin:cyclin dependent kinase (cyclin:Cdk) complex, controls cell cycle progression from yeast to mammals through the timed orchestration of substrate phosphorylation . Cell cyhp) of yeast and mammalian cyclins . The NLxxxL motif, DOC_CYCLIN_yClb5_NLxxxL_5, mediates substrate docking to S-phase cyclins Clb5/6  motif (LIG_ActinCP_CPI_1) and the twinfilin-type CPI motif (LIG_ActinCP_TwfCPI_2), affect actin polymerisation through direct binding of the heterodimeric actin capping protein (CP) that binds to and limits the polymerisation of the barbed ends of actin filaments. While binding of regulators through the canonical CPI motif allosterically down-regulates the capping activity of CP , the binSLiMs play prominent roles in regulated transport of cargoes between cell compartments by membrane vesicles and the maintenance of complex endomembrane systems Figure . The ESCLYPxL motifs bind the V-domain of Alix, another ESCRT system component involved in endosomal sorting of membrane proteins. Although our insights into the cellular roles of these motifs are rather limited, they are of prime importance because several viruses use them to hijack the ESCRT machinery for the budding of viral particles from the host cell membrane . In the This release also includes the classical adaptin-binding acidic dileucine motif (TRG_DiLeu_BaEn_1) and its five variants that almost exclusively occur on the cytosolic side of transmembrane proteins, frequently located near to the N- or C-termini. These motifs bind to a highly conserved site on the sigma subunits of clathrin-associated adaptin complexes (adaptins AP1-4) to initiate clathrin-mediated endocytosis or protein sorting to endosomes/lysosomes ,44. DileTheileria annulata  signalling and downregulation, endocytosis and cytoskeletal rearrangements, and the clustering and signalling of various other membrane receptors . Numerouannulata  and Toxoa gondii ) that emLiquid-liquid phase separation (LLPS) is a recently discovered, albeit fundamental molecular process that is driven by interactions between proteins and nucleic acids to form functionally specialised liquid compartments, the so called membraneless organelles, within cells . Phase-sSLiM-mediated interactions typically contribute to receptor clustering and the formation of the associated, membrane-proximal signalling networks , such as the synaptic densities of excitatory ,58 and iAmong the three novel motif classes functioning in LLPS processes, the LIG_DLG_GKlike_1 class collects phospholigands of the guanylate kinase-like (GK-like) domains of the discs large homologue (DLG) protein family that contains major scaffold proteins of postsynaptic densities (PSDs). The binding of PSD-95 to repeated phosphorylated motifs within SAP90 represents an essential link in the formation of PSDs ,73.PxpxPR motifs within the B-cell linker protein  belong to the newly annotated LIG_SH3_CIN85_PxpxPR_1 motif class and mediate multivalent interactions with CIN85 trimers, forming the extended molecular scaffold underlying phase-separated B-cell receptor signalling clusters .2 fixation, called the Pyrenoid. Formation of the Pyrenoid matrix depends upon multivalent, low-affinity interactions between RuBisCO binding motifs (LIG_RuBisCO_WRxxL_1) of the protein EPYC1 and the RuBisCO small subunit , mediating interaction with integrins, a class of human cell surface receptors known to be targeted by several other viruses . In addi-binding motif , providing a direct interface to endocytosis components. In addition, ACE2 also contains SLiMs capable of interacting with I-BAR domains (LIG_IBAR_NPY_1), PDZ domains (LIG_PDZ_Class_1) an endocytic sorting signal (TRG_ENDOCYTIC_2), and SH2 domains (SARS-CoV-2 hijacks native human SLiM-mediated interactions inside the host cell as well, modulating the endocytic and autophagy machinery . These S domains . Most of domains  regulatiToxoplasma gondii is an apicomplexan parasite that primarily infects cats and their prey but is also highly prevalent in the human population, being the causative agent of toxoplasmosis. All apicomplexans are intracellular parasites that invade host cells through the coordinated secretion of proteins contained in specialised organelles. Recent research has highlighted the role of SLiMs in host cell entry in Toxoplasma (T. gondii introduces itself into the host cell, creating the parasitophorous vacuole. These motif instances provide a link to cytoskeletal rearrangement through members of the ESCRT system and microtubule-binding proteins (Figure xoplasma , in parts Figure .In addition to sharing the motif annotation data, the ELM server also enables exploratory analyses of SLiMs on the protein sequences submitted by the user. The search results are accessible via graphical and tabular representations with details of the regular expression matches. The graphical view provides an overlay of motif match information with the sequence's domain, secondary structure, and other key contextual features. Regions predicted by GlobPlot  to be inWhile ELM provides a good starting point for motif exploration on a particular protein sequence, it is also expected that most regular expression matches are unlikely to be meaningful, especially for motif types that have low sequence complexity. Therefore, it is equally crucial to consider the biological context of the SLiM candidates. We have shared our recommendations to avoid typical mistakes during motif explorations . In our Advances in experimental and computational methodologies continue to be added to the toolkit for SLiM investigations. Deep mutational scanning enabled systematic evaluation of the contribution for all residue possibilities within the LP cyclin motif . The cryThe current update of the ELM database includes 30 novel SLiM classes, major revisions to five existing motif classes and a total of 411 new instances. New data developments in ELM have mainly focussed on cell cycle regulation, actin cytoskeleton regulation, membrane remodelling and sorting pathways, the formation of phase-separated liquid compartments and integrin signalling. These major areas of cell regulation are also heavily affected by pathogenic motif mimicry, therefore the current release of ELM not only captures information into how eukaryotic cells operate, but also into how they get efficiently invaded by various pathogens. In the coming years, the motif biology field and the ELM resource itself will need to tackle the challenge of integrating motif data as it becomes available from high-throughput motif discovery approaches , and the


BackgroundGroin reconstruction with muscle flap coverage is associated with high wound complication rates. Incisional vacuum-assisted closure (iVAC) therapy may lower wound complications. We evaluated the impact of iVAC on postoperative outcomes in patients following groin reconstruction with muscle flap coverage.MethodsWe conducted a retrospective review of patients who underwent groin reconstruction with muscle flap coverage in 2012-2018. Patients were divided into those who received iVAC therapy and those who received standard sterile dressings (SSD).Resultsn = 41) and the rest received SSD . The iVAC group had higher rates of diabetes, hypertension, coronary artery disease, and peripheral artery disease (p < 0.05). However, iVAC patients had comparable length of hospital stay (12 vs 8.5 days p = 0.0735), reoperations , and readmissions  with SSD patients. iVAC placement was less likely in prophylactic flaps .Of the 57 patients included, most received iVAC therapy (71%, ConclusionPatients with a prophylactic flap were less likely to receive vacuum therapy, which may highlight a selection bias where surgeons pre-emptively use iVAC therapy in surgical candidates identified as high risk. The pre-emptive use of iVAC may minimize adverse postoperative outcomes in high-risk patients. Wound complications after major surgery on the groin are a serious problem associated with significant morbidity and cost. Complication rates as high as 44% have been reported in infra-inguinal vascular procedures -3. SurgeSeveral methods have been proposed to reduce the complication rates in groin surgery. The use of muscle flaps to treat complications following vascular surgery on the groin was first described in 1989 by Mahoney et al. and has become a widely accepted method of salvage therapy -13. MoreAnother approach to complication rate reduction in groin surgery is with the use of vacuum-assisted incisional closure therapy (iVAC). The use of iVAC therapy is thought to provide benefits and decrease complication rates by acting as a barrier to the incision from external infectious sources, helping promote skin edge apposition, remove fluid and infectious materials from the incision, and increase microcirculation ,14-18. AThe purpose of this study was to delineate the utility of the regular use of iVACs in groin reconstruction via retrospective analysis of patients who have undergone groin reconstruction with muscle flap coverage.This project was approved by the University of Maryland Institutional Review Board. We performed a retrospective review of all patients who had received groin reconstruction from December 2012 to December 2018. Eligible patients were identified with current procedural terminology (CPT) codes for muscle flap coverage and groin reconstruction . An example case is presented in Figure Patients were divided into two groups: vacuum-assisted incisional closure (iVAC) Figure  and stanComposite data were stored in Microsoft Excel  and analyzed using the Statistical Package for the Social Sciences (SPSS) software version 26 . Non-parametric variables were summarized and analyzed using median values and interquartile ranges. Categorical data were summarized using percentages. Differences in continuous data between the groups were tested with the non-parametric Mann-Whitney-U test. The chi-squared test and Fisher exact test were used to test for differences between categorical data, as appropriate. The odds ratio (OR) is reported with 95% confidence intervals (95% CI). Variables found to be significantly different between the two groups in univariate analysis at p<0.25 and clinically relevant variables as determined by clinical judgment were included in the multinomial logistic regression. Variables were checked for multicollinearity  and were excluded if multicollinearity was found (VIF >1.5). Statistical significance was defined as a two-tailed value of p≤0.05.Fifty-seven patients were included in our study. Nearly three quarters received an iVAC 71%, n=41) and the rest received an SSD . The patient and surgical characteristics of the two study groups are summarized in Table %, n=41 aA greater proportion of patients in the iVAC group had a history of prior groin surgery as compared to the SSD group . Additionally, 78% of patients who underwent iVAC placement had salvage, rather than prophylactic, muscle flaps as compared to 19% of patients in the SSD group with salvage muscle flaps (p<0.0001). There were increased odds of iVAC use with salvage flaps . Groin reconstruction for infective indication was more common in patients who underwent iVAC placement than the SSD group , whereas oncologic reconstruction was significantly more prevalent in the SSD group . The most common muscle flaps utilized in the iVAC group were the sartorius , gracilis , and rectus femoris , whereas the SSD group almost exclusively received a gracilis muscle flap .The comparison of postoperative outcomes is shown in Table Based on univariate analysis and clinical judgment, we identified variables of interest for multinomial logistic regression. Infection as an operative indication was found to be collinear with salvage flaps (VIF = 2.27) so was excluded from the analysis. We assessed risk factors for surgical site complications, reoperations, and readmissions Table . The useThe aim of this study was to evaluate the utility of iVAC use in patients with muscle flap reconstruction of the groin. Our results show that iVAC therapy in groin reconstruction with muscle flap has comparable outcomes to standard sterile dressings despite use in a higher risk patient population. Furthermore, diabetes mellitus is the most important predictor for postoperative complications and reoperations within this patient population.In our patient population, patients who received iVAC therapy were more likely to have risk factors such as diabetes, hypertension, coronary artery disease, and peripheral artery disease. Our results demonstrate that diabetes was an independent risk factor for surgical site complications and 90-day reoperations, which is consistent with previous literature ,20-21. AAdditionally, patients were more likely to receive iVAC therapy in salvage groin reconstruction surgery, rather than prophylactically. The use of muscle flap as salvage therapy in wound complications of the groin has a well-studied benefit due to increased vascularization of the complication site, elimination of wound dead space, and increased lymphatic drainage around the site of transposition -10,12-13Multivariate analysis demonstrated that iVAC did not influence surgical site complications, 90-day reoperations, or 90-day readmissions. This calls into question whether iVAC, which can cost up to $495, conveys a significant enough benefit to outweigh the inexpensive standard sterile dressing . FurtherThere are several limitations to our study. A major limitation is the introduction of selection bias, as patients were not randomized to iVAC vs SSD and the use of iVAC-assisted therapy was based on the surgeon’s discretion. Patients who were at higher preoperative risk were more likely to receive iVAC therapy. This is evidenced by the higher rates of diabetes, coronary artery disease, peripheral artery disease, and hypertension in the iVAC group. These factors may account for comparable outcomes between the iVAC and SSD groups. Patients with a prophylactic flap were less likely to receive iVAC therapy, which may further highlight selection bias in which surgeons pre-emptively use iVAC therapy in patients with pre-existing wound complications. This study also included patients treated by several different surgeons in varying surgical specialties, including general surgery, plastic surgery, and vascular surgery. Thus, inter-operator variability may be a confounding factor that was not accounted for in our analysis.Patients who received iVAC therapy had comparable complication, reoperation, and readmission rates to those who received standard dressings despite the possible higher preoperative risk for complications amongst the iVAC therapy group. The use of vacuum-assisted incisional closure therapy may be linked to a selection bias by the surgeon and used preferentially in patients perceived to be at high risk for complications after muscle flap groin reconstruction. The pre-emptive use of iVAC may minimize adverse postoperative outcomes in high-risk patients.


During a seizure, there is a powerful and forceful contraction of muscles which may lead to fractures or joint dislocations. However, multiple periprosthetic hip fractures and joint dislocations secondary to seizures have not been reported.A 49-year-old male developed spontaneous and bilateral multiple periprosthetic hip fractures and joint dislocations  secondary to generalized convulsive seizures which occurred within few hours after bilateral total hip arthroplasties (THAs). Bilateral open reconstruction and fixation were performed on the 21st day after primary THAs and on 2-year follow-up, the patient showed satisfactory functional outcome.Multiple periprosthetic hip fractures and joint dislocations secondary to seizure are extremely rare, and treatment targets for these injuries should focus on fracture healing and limb function recovery. Craniocerebral operation could bring an elevated risk of seizure; meanwhile, subsequent corticosteroid replacement threapy was complicated by secondary osteoporosis. Therefore, anti-osteoporotic and anti-epileptic therapy should be considered in this type of patients to avoid fracture and dislocation after arthroplasty. Although aseptic loosening, instability, osteolysis and infection are common reasons for revision after total hip arthroplasty (THA), malfunction of endoprostheses also can be caused by periprosthetic fracture (PPF) . PatientHere we present a case of simultaneously bilateral multiple periprosthetic hip fractures and joint dislocations  in a 49-year-old male following a generalized convulsive seizure in view of its rarity and favorable prognosis after appropriate management. Meanwhile, we also described the occurrence mechanism of these injuries, as well as the experience and deficiencies in the treatment of this patient. It may increase the awareness of such injuries among emergency physicians, neurologists, and orthopedic surgeons. We present the following article in accordance with the CARE reporting checklist.A 49-year-old male was referred to the orthopedic department of a tertiary hospital with pain in both hips for 19 months and the X-ray examination showed bilateral femoral head collapse and secondary osteoarthritis  . Before any further intervention was applied, another episode of generalized convulsive seizure with severe extremity contractions occurred 4 h after the first one. There was no evidence of open wound, falling from bed, external trauma, bruising or distal neurovascular deficits during seizure episode and afterward. The patient was not a known epileptic before.Associated symptoms being controlled, this patient complained of severe pain and immobility of both hips. Meanwhile, both lower limbs were externally rotated and markedly foreshortened on clinical examination. Plain radiographs and CT of the hip joints showed backward and upward dislocation of the right femoral head, displaced oblique fracture of the proximal right femur involving greater trochanter, upward dislocation of the left hip joint involving acetabular endoprosthesis components, fracture of the left acetabular and avulsion fracture of left lesser trochanter Fig.  e & f.FSince the patient was vulnerable after the last operation, a bilateral revision of total hip arthroplasty was performed in one session on the 21st day after THA when the patient’s condition has stabilized, with anti-epileptic drugs and prednisolone continuing to be administered before revision. The posterolateral approach was followed on both sides. The right hip surgery was performed first: after removal of the femoral prosthesis, fracture flats of the proximal femur were restored and fastened with three titanium cables; then a new set of non-cemented long-stem prosthesis (DePuy) and 36-mm ceramic head was installed, and the dislocated hip joint was reducted. As to the left hip joint, since the superior acetabular bone was defective, an allogeneic femoral head was trimmed and fixed to the region of bone loss by using three cannulated screws. Acetabulum being carefully polished, a new porous 54-mm PINNACLE acetabular cup (Depuy) was placed and fixed with 4 cancellous bone screws; then a 36-mm highly cross-linked polyethylene lining was embedded and dislocated femoral head was reducted.After the operation, the patient was shifted to the intensive care unit and closely monitored for the first 24 h. The postoperative practices including straight-leg elevation, ankle pump and quadriceps isometric contraction were started on the 1st day after surgery and the patient began progressive bedside standing and ambulating.2 weeks later. On 6-month follow-up, there was a good union of fractures both clinically and radiologically Fig. a, b & c;Periprosthetic fracture is one of the toughest problems after total hip arthroplasty, which often occurs following a high energy trauma or secondary to any indirect mechanisms that weaken surrounding bones like osteomalacia and osteoporosis , 9. InjuPostoperative PFF, which generally happens following a low-energy fall from standing height, shows a close relationship with stem loosening and poor clinical outcomes , 11. BonDislocation of bilateral joints was due to the powerful and violent contraction of hip muscle during convulsion which generated a force directing proximal femur towards the groin , 19. ForSome lessons could be learned from this case. Surgical stress and a history of former craniocerebral operation both act as incentives for this epileptic attack. Other possible etiologies for seizures such as hypocalcemia, hypoglycemia and dyselectrolytemia were excluded by using routine biochemical investigations. It was negligence that anti-epileptic therapy was not administrated before arthroplasty; which was partly because the patient had never had an epileptic seizure before. After the first attack of seizure, although associated examinations were performed, anti-epileptic medication was not applied immediately and the occurrence of the second seizure induced this terrible problem. Another negligence was that we did not attempt to determine bone density/osteoporosis prior to the time of surgery considering the patient’s age and body condition. This patient was admitted for glucocorticoid-induced ONFH, which was the most common type of non-traumatic ONFH. Long-term administration of antiepileptic medications and corticosteroid may also cause anticonvulsant osteoporosis and therefore act as a risk factor for epilepsy-induced fracture . There wBilateral periprosthetic hip fractures and joint dislocations induced by seizure are life-threatening injuries. Treatment targets for periprosthetic hip fractures and joint dislocations should focus on fracture healing and limb function recovery. The craniocerebral operation could bring an elevated risk of seizure and subsequent corticosteroid use was associated with secondary osteoporosis. Therefore, anti-osteoporotic and anti-epileptic therapy should be considered in this type of patients to avoid fracture and dislocation after arthroplasty.


Viral hepatitis is one of the main causes leading to hepatocellular carcinoma (HCC). The continued rise in incidence of HCC suggests additional factors following infection may be involved. This review examines recent studies investigating the molecular mechanisms of chronic hepatitis and its association with hepatocarcinogenesis. Hepatitis B virus patients with genotype C display an aggressive disease course leading to HCC more than other genotypes. Furthermore, hepatitis B excretory antigen (HBeAg) seems to be a more sensitive predictive tumor marker exhibiting a six-fold higher relative risk in patients with positive HBsAg and HBeAg than those with HBsAg only. Single or combined mutations of viral genome can predict HCC development in up to 80% of patients. Several mutations in HBx-gene are related with higher HCC incidence. Overexpression of the core protein in HCV leads to hepatocellular lipid accumulation associated with oncogenesis. Reduced number and decreased functionality of natural killer cells in chronic HCV individuals dysregulate their surveillance function in tumor and viral cells resulting in HCC. Furthermore, high T-cell immunoglobulin and mucin 3 levels supress CD8+ T-cells, which lead to immunological dysregulation. Hepatitis D promotes HCC development indirectly via modifications to innate immunity, epigenetic alterations and production of reactive oxygen species with the LHDAg being the most highly associated with HCC development. Summarizing the results, HBV and HCV infection represent the most associated forms of viral hepatitis causing HCC. Further studies are warranted to further improve the prediction of high-risk patients and development of targeted therapeutics preventing the transition from hepatic inflammation–fibrosis to cancer. Hepatocellular carcinoma (HCC) represents the fourth most common cause of cancer-related death worldwide and is responsible for over 80% of primary liver disease ,2. VarioThe pathophysiology of HCC is a complex multistep process. Identification of the cell of origin remains debatable if arising from a hepatic stem cell or transformation of mature hepatocytes. A plethora of studies based on mouse models have described carcinogenesis originating from mature hepatocytes . Cancer-nduction . The exaHAV is a small non-enveloped virus, belonging to the Picornaviridae family . The virConsidering any potential direct impact on HCC, HAV lacks chronicity and progression to cirrhosis, which are thought to be prerequisites of hepatocellular oncogenesis  and HAV The HAV-related tissue injury is mediated by an intense immune response, rather than a direct HAV-related toxicity, and an uncontrolled latent stimulation of the host’s defence could be the main mechanism. Several reports have concluded that the presence of the HLA-DRB1*1301 allele is associated with slow HAV clearance, persistent liver damage and autoantibodies expression among patients with hepatitis A . In thisAdditionally, the impact of acute HAV infection in patients’ outcome is strongly dependent on the health of the liver at the time of infection. Thus, in patients with CLD, the addition of HAV infection predisposes to acute liver failure and/or cirrhosis decompensation and progress .Increasing evidence supports a unique and robust (auto-)immunity against liver antigens after HAV infection in certain populations. More specifically, patients with functional defects in T cells, regulating immune responses against specific surface liver antigens, were found to be vulnerable to type I AIH development after hepatitis A infection, due to persistence of upregulated T helper cells and antibodies against autoantigens . FurtherTaking together, HAV infection may exert a potential impact on HCC onset, progression and outcome, through immune dysregulation, and thus further large-scale studies are essential to illuminate in depth this field.Hepatitis B virus (HBV) is one of the most common chronic infections and represents a global health problem. With an estimated number of 257 million infected subjects worldwide, it represents the leading cause of developing HCC worldwide . The risEpidemiological studies exhibit evidence of a causal role of chronic HBV infection and the development of a HCC. Questions remain as to the exact molecular mechanisms by which chronic HBV infection leads to HCC. Hepatitis B virus is a partially double stranded DNA-virus with at least eight major genotypes (A to H). During an infection the HBV DNA is being integrated, transcribed and translated leading to reproduction of the virus and its components causing an inflammatory cascade within the hepatocyte. Repeated inflammatory cycles can lead to chronic inflammation and cirrhosis, which is a high-risk factor for hepatocarcinogenesis . NeverthChronic HBV infection represents the major etiological factor for HCC worldwide with more than one half of HCC patients being chronic carriers with an increased lifetime risk 25–37 times higher than non-infected patients . DevelopIn a prospective cohort study with more than 3600 patients with a mean follow-up of 11 years, it was demonstrated that the risk of HCC development was associated with high HBV DNA serum levels . In anotOn molecular level, HBsAg-positivity is generally associated in screening and epidemiological studies. Nevertheless, several case-control studies indicate that HBeAg is a better predictive marker for HBV-related HCC with higher prevalence. The relative risk is increased six-fold among patients positive for HBsAg and HBeAg than those positive for HBsAg only . Yang etin (AFP) .A possible correlation between BCP mutation and genotype C HBV was already mentioned. Independently from genotypes B and C HBV, the so-called “T1762/A1764” mutation in the basal core promoter (BCP) region has a high prevalence with HCC development in both genotypes . It can The HBx-protein is an activator for several host cellular genes responsible for DNA repair and growth control. Among the viral products, HBx protein has been termed “viral oncoprotein” by many authors due to its pleiotropic activities on cell cycle regulation, signalling pathways and DNA-repair . AffectiIn summary, chronic HBV infection represents the main risk factor for viral induced HCC development. Chronic HBV infection is responsible for malignant disease course even in absence of liver cirrhosis. While genotype C HBV is associated with Basal core promoter mutations, there is evidence that this factor might act independently in carcinogenic pathways. Furthermore, in patients with genotype C HBV there is evidence of higher HBeAg levels, which may explain the aggressive disease course. A substantial role in carcinogenic pathways are single and combined mutations in the core promoter region as well as in the Hbx-gene, which we outlined in this review. A recent meta-analysis underlined the PreS deletion being one of the most common mutations in HBV-related carcinomas among Asians . AlthougWorldwide, more than 170 million individuals are chronically infected with HCV . HCV canHCV infection results from a series of events involving translocation of blood-borne viral particles entering hepatocytes using a variety of cell membrane proteins as anchors for attachment with subsequent adjacent cell to cell transmission ,70. It ifibrosis . Of notefibrosis .The correlation between HCV infection and the development of HCC is well established . PreviouChronic HCV infection is strongly associated with the accumulation of lipid within hepatocytes . VariousImmune cell activation is instrumental in the early stages of HCV infection . ActivatIn a recent small study, Song et al. evaluated the immune cell phenotype within HCC tumor tissue. Their results highlighted changes in both macrophage and T cell populations as well as chemokine and cytokine patterns within tumor tissue which could alter HCC progression . SpecifiTumor surveillance is the function of a number of specialized immune cells including natural killer (NK) cells . Cell–ceNK cells . ChronicNK cells may also participate in the destruction of antigen specific T cell response where increased TIM-3 and PD-1 enhances NK cell engagement with and killing of helper T cells . As dischenotype . TogetheDAA therapy has proven remarkably successful as a curative agent in HCV infected individuals . While sIn summary, HCV infection promotes a wide range of changes within the liver, from lipid accumulation within hepatocytes to architectural changes associated with fibrogenesis which ultimately impact liver function. Central to these changes are oxidant stress and overall tissue damage which are known to predispose the liver to cancer development independent of HCV infection itself. Growing evidence demonstrates a wide variety of HCV associated alterations to immune cell responses, whereby recruitment, differentiation, and regulation are disrupted.Hepatitis D virus (HDV) was first described in 1977 and represents the sole member of the Deltavirus genus ,116,117.n = 299) with HDV/HBV co-infection, the annual incidence of HCC-onset was estimated to be 2.8% (follow-up for 19 years) [n = 200), it was demonstrated that HDV-infected cirrhotic patients exhibited three times more elevated risk for development of HCC [The role of HDV infection in liver oncogenesis remains a topic of debate . Despite9 years) . In a fut of HCC .n = 605). Two-thirds of them were regarded as having been inoculated with the virus via intravenous administration through drug use and another considerable percentage attributed nosocomially. The majority of the aforementioned HDV patients exhibited liver cirrhosis (77%) with 16.5% also having HCC. However, a statistically significant difference for HCC onset was not observed between HDV-HBV coinfection and mono-infection HBV patients, despite the more frequent decompensated liver cirrhosis of the former group [The Swiss HIV cohort reported that individuals with triple infection HBV/HDV/HIV who were followed-up for a median time of 8.7 years revealed a nine-fold and eight-fold increase in HCC risk and liver related death, respectively, . Relevaner group . In Gamber group .At the molecular level, since HDV does not integrate into the human genome, a direct oncogenic effect is unlikely . Howeverormation . Augmenttastasis . Likewistastasis . Activatogenesis . The L-Hpoptosis . Finallypoptosis . In thispoptosis .Hepatitis E virus (HEV) is listed among the various enteric viruses involved in food-borne outbreaks and regarded as the most common etiology of acute hepatitis world-wide . The nomThe clinical spectrum of HEV infection ranges from asymptomatic or acute cases to chronic hepatitis in immunocompromised patients. In patients with CLD, HEV may result in decompensation and death . HEV serViewing the aforementioned data, HEV appears to join along with HBV and HCV as a possible HCC etiology in chronically infected individuals. HEV may trigger HCC, as in the case of all other viral agents through the induction of chronic hepatitis . As withOver 90% of HCC cases occur in the setting of chronic liver disease. Cirrhosis from any etiology is the strongest risk factor for HCC ,148, whiIn contrast to HBV, no evidence of genotype-dependent factor is seen within HCV–HCC paradigm, although its viral clearance response might differ due to the HCV genotype . It seemCurrent studies were reviewed to emphasize factors responsible for HCC in viral hepatitis. Cirrhosis regardless of etiology remains the main risk factor. Molecular pathways in chronic HBV infection are well known and yet still their mechanism is not well understood. In addition, the viral genotype, numerous mutations in Basal Core promoter and HBx protein correlate with higher HCC incidences. Detection of these mutations may lead to early diagnosis and prevention of malignant courses. Co-infections with HEV or HDV accelerate disease progression and lead consequently to HCC. Viral factors in malignant pathways in chronic HCV disease are less described. The studies analysed herein focus mainly on host factors. Oxidative stress, altered immune cell response and the overall tissue damage play major roles in the HCV-HCC paradigm. While HBV/HDV co-infections lead to higher risk of cirrhosis, genotype 1 HDV had worse clinical outcome than genotype 2 including malignant course. HAV and HEV can lead to secondary diseases such as AIH or co-infections characterizing risk factors for malignant outcomes.It is clear from the preceding review that viral hepatitis represents an important predisposing factor for the development of HCC. Commonalities exist in the development of HCC following viral infection including chronic inflammation, epithelial to mesenchymal transition, and overt fibrosis and cirrhosis. Not all cases produce HCC within a given hepatitis virus type, suggesting that additional factors may be at play, including host responses and secondary pathologies including metabolic syndrome and fatty liver disease. Indeed, the strong correlation among fibrosis progression and HCC development highlights the need for additional drivers of disease progression. Moreover, HCC incidence remains elevated following therapeutic clearance of virus further implicating additional factors in its occurrence. Indeed, emerging evidence indicates prolonged alterations in regulatory immune cell processes which may enhance the initial inflammatory response as well as impair secondary surveillance pathways critical for clearance of altered parenchymal cells within this inflammatory microenvironment both acutely as well as chronically. Future studies aimed at understanding the impact of various virus types on the host immune cell response may provide new insights into HCC occurence in these patients as well as new therapeutic targets to limit or prevent this disease progression.


Primary squamous cell carcinoma (SCC) of stomach is extremely rare. The pathogenesis of SCC of stomach remains unclear. There is only one report that Epstein Barr virus (EBV) infection may be involved in the pathogenesis of SCC arising in the stomach ever before. Here, we report a case of Epstein Barr virus infection-associated primary SCC of stomach in a 70-year-old woman. She was presented to the referring hospital with hematemesis. Initial endoscopy revealed a bleeding gastric ulcer in the upper part of gastric corpus and the coagulation therapy was followed. After a 3-month follow-up, endoscopy revealed a submucosal tumor-like protrusion instead of an ulcer. Computed tomography revealed a mass in the upper part of stomach and swollen lymph nodes along with the lesser curvature and para-aortic lymph node. Biopsy could not confirm the definitive diagnosis. We performed total gastrectomy with para-aortic lymph node sampling. Histological analysis revealed squamous cell carcinoma with EBV infection with lymph node metastases. Tumor cells were positive for EBV-encoded small RNA (EBER) by in situ hybridization. The postoperative course was uneventful and the patient was discharged on day 11 after the operation. CapeOX was started as adjuvant chemotherapy, and the patient remains alive without recurrence 7 months after surgery.This is the first case report of EBV infection-associated primary SCC of the stomach diagnosed by in situ hybridization of EBER. EBV infection may be related to the pathogenesis of primary SCC. Further evidence and studies are required to establish optimal strategy for this rare disease. Primary squamous cell carcinoma (SCC) of stomach is extremely rare –3. To da[18]F-FDG PET/CT imaging demonstrated strong FDG uptake in a mass of stomach and the lymph nodes of lesser curvature, otherwise uptake of FDG in para-aortic lymph node was weak  revealed a bleeding gastric ulcer in the upper part of gastric corpus Fig. A and theeak Fig. B. BiopsyThe macroscopic examination of the resected specimen revealed a protruding mass, measuring 38 × 24 mm, in the gastric cardia along the posterior wall  allThe immunohistochemistry of CK5/6 and p40  is also Primary SCC has an aggressive behavior, such as metastasizes to lymph node, liver and other organs, they are usually diagnosed at advanced stage, which leads to poor prognosis –13. The Although the pathogenesis of the primary SCC of stomach is still unknown, several hypotheses concerning the origin of the SCC have been reported as follows , 15, 16:EBV infection is known to cause gastric cancer, and EBV infection-associated gastric cancer accounts for approximately 10% of all gastric cancer . EBV infA histological feature of EBV infection-associated gastric cancer is a mainly diffuse-type carcinoma accompanied by abundant lymphocyte infiltration. In case of the tumor invading submucosa, undifferentiated cancer cells and infiltrated lymphocytes compose a mass, causing submucosal nodules .Recent cancer genome atlas research has divided gastric cancer into four molecular subtypes as follows : (1) tumIn our case, histopathological findings revealed the partially keratinization in the tumor cells without gland formation or mucus production, indicating poorly differentiated SCC. Also immunohistochemistry staining of p40 and CK5/6 was positive. There was no squamous metaplasia or ectopic squamous epithelium around the tumor. Tumor cells invaded into subserosa and infiltration of lymphocytes and plasma cells into the stroma was found. All tumor cells were positive for EBER by in situ hybridization. Therefore, we diagnosed this case as EBV infection-associated primary squamous cell carcinoma of stomach. EBV infection-associated primary SCC of the stomach is very rare, and to our knowledge, there have been no other reports ever before which revealed the EBV infection using in situ hybridization. We performed radical resection, but considering lymph node metastasis in resected specimen, adjuvant chemotherapy was required. A standard chemotherapy regimen for this disease has not been established; the choice of its treatment tends to follow the principle of the treatment against gastric adenocarcinoma. On the basis of adjuvant regimen of gastric adenocarcinoma and the regimen against SCC of esophagus, we selected CapeOX. EBV infection-associated gastric cancer has molecular biological characteristics such as DNA methylation and PD-L1/2 overexpression, demethylating agents or anti-PD-L1 antibody may be one of the treatment options in the future. We need further research in this tumor and further studies would benefit the patients affected by this rare disease.In conclusion, here we described a case of EBV infection-associated primary SCC of stomach. Adjuvant chemotherapy following radical resection leads to favorable outcomes. Further evidence and studies are required to establish optimal strategy for EBV infection-associated primary SCC of the stomach.


The field of cellular microbiology, rooted in the co-evolution of microbes and their hosts, studies intracellular pathogens and their manipulation of host cell machinery. In this review, we highlight emerging technologies and infection models that recently promoted opportunities in cellular microbiology. We overview the explosion of microscopy techniques and how they reveal unprecedented detail at the host-pathogen interface. We discuss the incorporation of robotics and artificial intelligence to image-based screening modalities, biochemical mapping approaches, as well as dual RNA-sequencing techniques. Finally, we describe chips, organoids and animal models used to dissect biophysical and in vivo aspects of the infection process. As our knowledge of the infected cell improves, cellular microbiology holds great promise for development of anti-infective strategies with translational applications in human health. Cellular microbiology is a field that combines the study of infection and cell biology. In this review, we highlight emerging technologies and infection models that recently transformed our understanding of the infected cell and may inspire future medicine. Listeria and Shigella, are central to the field of cellular microbiology  and one of the best examples of infection biology contributing to fundamental cellular processes comes from the reconstitution of bacterial ‘actin tails’ .Bacterium-containing vacuole (BCV): intracellular vesicular compartment that results from the internalisation of bacteria into host cells.Cell-autonomous immunity: ability of immune and non-immune cells to protect themselves against intracellular bacterial pathogens using highly conserved defence pathways .Click chemistry: bioorthogonal reactions in which small biocompatible reporters are conjugated to biomolecules of interest, used in various live-cell applications (including photoactivable fluorogenic labelling of proteins and lipids).CRISPR libraries: collection of single guide RNA (sgRNA) molecules for subsequent CRISPR/Cas-9 gene editing (CRISPR knockout) or transcriptional regulation .Deubiquitinase: protease that cleaves ubiquitin from ubiquitinated proteins for their localisation, activity and dynamics.Diffraction limit: minimum angular separation between two objects that can be distinguished by an optical microscope.Galectins: type of lectins or carbohydrate-binding proteins with affinity for β-galactosides, used as a marker for membrane damage at the BCV as it binds to luminal glycans when exposed to the cytosol.Guanylate-binding proteins (GBPs): family of IFN-inducible GTPases that target host membranes and bacterial surfaces to regulate inflammasome activity and antibacterial defence mechanisms.High-content screenings: phenotypic screenings focused on the complex analysis of multiple parameters .High-throughput screenings: phenotypic screening focused on the rapid analysis of multiple candidates, normally based on a single parameter (such as the growth of intracellular bacteria during multiple drug treatments).Inflammasome: multiprotein complex that mediates secretion of pro-inflammatory cytokines and programmed cell death.Macropinosomes: vesicles produced as a result of macropinocytosis, a type of endocytosis that involves non-specific uptake of extracellular material.M cells: specialised epithelial cells in the intestine important for host defence.Microfluidic chips: device containing micro-chambers, channels and inlet/outlet ports that enable analysis of single cells by microscopy.Neutrophil extracellular traps (NETs): networks (composed of DNA and proteins) with antimicrobial activity that are secreted by neutrophils in response to extracellular pathogens.Nuclear Factor-κB (NF-κB): protein complex that regulates transcription of immune factors and pro-inflammatory molecules (such as cytokines) in response to infection.NOD-like receptors (NLRs): highly conserved pattern recognition receptors (PRRs) that recognise cytosolic danger signals for cell-autonomous immunity.Organoids: self-organised 3D tissues derived from stem or progenitor cells, which resemble a specific organ.Peyer’s patches: specialised lymphatic tissue present in the small intestine organised as follicles.Phenotypic heterogeneity: in the case of bacterial cells, the presence of different phenotypes (e.g. susceptibility to antibiotic treatment) that naturally occurs within an isogenic population.Post-translational modifications: covalent modification of proteins that enables their modulation  in response to environmental stimuli.Septins: group of GTP-binding cytoskeletal proteins that assemble to form filaments, bundles, rings and cage-like structures.Tolerance: host mechanism during infection that limits pathology (but does not restrict the pathogen) and promotes survival.Type III Secretion System (T3SS): secretion machinery found in Gram-negative bacteria, essential for invasion and pathogenicity of enteric bacteria (such as S. Typhimurium and S. flexneri).Type VI Secretion System (T6SS): secretion machinery found in Gram-negative bacteria (such as V. cholerae and P. aeruginosa), essential for mediating bacterial interactions during competition.Type VII Secretion System (T7SS): secretion machinery found in Gram-positive bacteria (such as L. monocytogenes and S. aureus) and mycobacteria (such as M. tuberculosis), playing important roles in bacterial physiology, competition and virulence.Ubiquitination: post-translational modification in which a protein or lipid is covalently linked with one or more ubiquitin residues, in a three step enzymatic process involving a ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin-ligase (E3).Standard widefield and confocal microscopy techniques historically provided the scale and resolution required to enable major discoveries in cellular microbiology, such as mechanisms underlying the internalisation and actin-based motility of invasive bacterial pathogens. In this section, we highlight state-of-the-art microscopy techniques providing unprecedented opportunities to investigate bacteria–host cell interactions Fig. .Fig. 1St3). Super-resolution microscopy comprises an expanding family of techniques with a common characteristic: their ability to surpass the ‘diffraction limit’. These include structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED) and single-molecule switching nanoscopy (SMSN), which have been developed concomitantly with improved dyes, probes and labelling methods4. Super-resolution microscopy is instrumental to focus on individual molecules and access previously unresolvable subcellular structures, such as the bacterial cytoskeleton, cell wall and nucleoid, as well as the highly structured and compartimentalised bacterial cytoplasm (reviewed in Singh and Kenney5). SMSN methods are based on photoswitchable molecules, so that their emission signals are spatiotemporally separated and signal centres can be determined with nm resolution. As a consequence, SMSN can reveal detailed insights into the architecture, stoichiometry and localisation of subcellular structures6. While originally these techniques had limited axial resolution (2D SMSN), the use of dual detectors with ‘4Pi geometry’ increased the localisation precision in z (3D or 4Pi-SMSN)6. Both 2D and 3D SMSN were used to investigate the ‘Type III Secretion System (T3SS)’ of Salmonella enterica serovar Typhimurium7, an important zoonotic pathogen causing gastroenteritis and inflammation of the intestinal mucosa. Although the T3SS needle complex has been isolated and studied in depth, the organisation of the entire T3SS (including the sorting platform) was less understood. In a recent study using SMSN, authors revealed the ultrastructure, protein stoichiometry, assembly and distribution of the T3SS in live bacterial cells, as well as the localisation of bacterial effectors prior to secretion7. Super-resolution microscopy offered fundamental insights into the secretion of bacterial effectors and growth of bacteria in broth, and in the future can be used to study bacterial pathogens inside cells. However, this remains challenging, considering that super-resolution microscopy is sometimes laborious  and often requires high laser intensities incompatible with living samples. The continuous improvements to super-resolution microscopy  will increase the number of studies exploring single-cell bacteria physiology during infection and reveal further adaptations to different intracellular niches.An important breakthrough in the field of fluorescence microscopy was the invention of super-resolution microscopy ’, a small percentage of bacteria rupture the BCV and escape to the cytosol where they hyper-replicate. However, to prevent hyper-replication, the luminal leaflet of the broken BCV is recognised by ‘galectins’8, and the LPS of cytosolic bacteria become ‘ubiquitinated’10. These events recruit selective autophagy cargo receptors, including p62/SQSTM1, NDP52/CALCOCO2 and OPTN, that target Salmonella to degradation by autophagy. While standard confocal microscopy provided evidence that autophagy cargo receptors arrange in distinct microdomains at the surface of Salmonella11, the precise arrangement of ubiquitin on the surface of S. Typhimurium was uncovered by direct Stochastic Optical Reconstruction Microscopy (dSTORM)12. In this study, the ‘deubiquitinase’ OTULIN was shown to restrict bacterial growth by controlling the density of linear ubiquitin chains on the bacterial surface, which serves as a platform for NF-κB activation.While 13. Here, multiple thin sheets of light illuminate the specimen  providing fast imaging with high axial resolution and minimal photobleaching. The application of LLSM has been limited by the complexity of set-ups being custom-built, and also by the requirement of computer-intensive postprocessing procedures. Consistent with this, LLSM has not yet been used to study infection. However, mitochondria can be viewed as ancient bacterial cells, and a study using LLSM showed that mitochondria make actin tails similar to those of Rickettsia (an obligate intracellular pathogen and α-proteobacteria closely-related to mitochondria)14. The recent release of commercial LLSM will undoubtably expand its use in infection biology.Finally, lattice light-sheet microscopy (LLSM) is a newly developed super-resolution microscopy method, which capitalises on previous microscopy techniques  and provides gentle and fast super-resolution in a volumetric sample15. Expansion microscopy can be used on its own, or its resolution can be enhanced when used in combination with conventional super-resolution microscopy techniques as demonstrated by the study of eukaryotic structures16. Its application to bacteria has great potential to provide nm-scale insights but it cannot be applied to live samples, and careful precautions are required to ensure that expansion does not affect the localisation or ultrastructure of complexes of interest. Although the expansion of bacteria has been technically challenging considering the rigidity of prokaryotic cell walls, digestion of the cell wall with enzymes (such as lysozyme) has been used17. In this study, the authors detected bacterial cell wall damage in untreated S. Typhimurium (as cells expanded) during infection of mouse RAW264.7 macrophages. Interestingly, the damage did not occur in all intracellular bacteria homogenously and varied for different bacteria inside the same macrophage. This lack of uniformity in an isogenic population, called ‘phenotypic heterogeneity’, remains a major challenge for effective antibiotic treatment18. Recently, a functionalized ceramide has been developed to simultaneously enable fluorescence labelling with Dibenzocyclooctyne (DBCO)-containing dyes (via ‘click chemistry’)19 and chemical fixation (with the addition of a primary amine), thus enabling visualisation of lipids during expansion microscopy20. This modified ceramide can integrate into membranes of pathogenic bacteria known to exploit host sphingolipids, including Neisseria gonorrhoeae and Chlamydia trachomatis. In this study, the combined use of click-labelled fluorescent ceramide, expansion microscopy and SIM enabled, for the first time using light microscopy, visualisation of the separated inner and outer membrane of Gram-negative bacteria20.In the absence of sophisticated optics, a resolution higher than offered by conventional fluorescence microscopy can be achieved using expansion microscopy. Here, the sample is cross-linked to a polyelectrolyte hydrogel which has the capacity to swell when immersed in water. This enables physical expansion of the biological sample (up to 100 times), whose molecules separate in an isotropic manner21), including BCV membrane rupture caused by several species of mycobacteria23, which enables bacterial escape to the cytosol and is dependent on the mycobacterial ESX-1 ‘Type VII Secretion System (T7SS)’. Similar to TEM, the full potential of FIB-SEM can be achieved in combination with correlative light and electron microscopy (CLEM), in which ultrastructural information (achieved by electron microscopy) is combined with molecular specificity and positional information provided by a fluorescent marker . Correlative FIB-SEM has been used to visualise the invasion of Shigella flexneri in HeLa cells. Here, the S. flexneri BCV was found to be closely surrounded by Rab11 positive ‘macropinosomes’, recruited via bacterial effectors IpgD, IpgB1 and IpgE, which determined the timing of BCV rupture24. A separate study showed that S. flexneri ipgD mutants are enclosed in a dense actin structure, called a cocoon, proposed to delay bacterial escape to the cytosol25. Brucella is a vacuolar pathogen that resides in a compartment, which first undergoes phagosomal maturation  and then acquires endoplasmic reticulum (ER) characteristics to support bacterial replication (replicative BCV)26. Correlative SIM and FIB-SEM revealed that membranes of replicative BCVs form a complex and continuous network with ER cisternae and nuclear membrane, and also showed fusion events between different BCVs26. In addition to long acquisition times , one limitation of correlative FIB-SEM studies is that organelle segmentation is often performed manually, which is time-consuming and data tends to be mostly descriptive. Significant efforts are being advanced in the field of computer vision and artificial intelligence for automated segmentation of 3D electron microscopy images27, which will reveal architectural features of the infected cell and provide a quantitative aspect to these studies. Another major drawback of electron microscopy techniques such as TEM, SEM or FIB-SEM, is that the preparation of biological samples requires the chemical fixation, dehydration and embedding of the specimen in resin, which can lead to severe artifacts. However, these limitations were overcome with the invention of cryo-electron tomography (cryo-ET). In this case, the sample is rapidly plunged into liquid ethane at ~80 K and becomes vitrified in its hydrated native state. Cryo-ET allows visualisation of any biological structure in its cellular context with a resolution of <5 nm. Although any bacterial structure is potentially amenable to this technique, cryo-ET has been instrumental to capture ultrastructure of different bacterial secretion systems including the Type II Secretion System (T2SS) and Dot/Icm Type IV Secretion System (T4SS) of Legionella pneumophila, as well as the ‘Type VI Secretion System (T6SS)’ of Vibrio cholerae (reviewed in Oikonomou and Jensen28). This information has enabled their structural and evolutionary comparison, providing a detailed understanding of their composition, integration in the bacterial membrane and mechanism of action. Cryo-ET has also been used to explore the precise arrangement of host cell actin during the invasion of S. Typhimurium in HeLa cells, which discovered different invasion mechanisms dependent on the Salmonella effector SipA29. Correlative light and cryo-ET has been exploited to visualise the precise arrangement of ‘septins’ surrounding S. flexneri and Mycobacterium smegmatis, which (as observed in vitro) assemble at the bacterial cell pole as filaments  to entrap bacteria in cage-like structures30.For many decades, transmission and scanning electron microscopy techniques  enabled ultrastructural inspection of infected samples. However, a general limitation of these techniques is the acquisition of 3D information, which can be laborious . This limitation was overcome ~10 years ago with the innovation of focused ion beam scanning electron microscopy (FIB-SEM). FIB-SEM combines the serial layered ion milling of a volumetric sample with the orthogonal acquisition of exposed surfaces, to obtain a spatial resolution of ~20 nm in z. For this reason, FIB-SEM is optimal to analyse the 3D morphology of organelles and their association with subcellular structures. Shortly after its innovation, FIB-SEM was applied to investigate the manipulation of host cell organelles by various intracellular pathogens 32. Here, the authors could trace the presence of bedaquiline (a lipophilic antitubercular compound) because of its bromine atom. Bedaquiline was shown to accumulate in macrophage lipid droplets, where it is progressively transferred to M. tuberculosis during infection. The discovery that lipid droplets can serve as a reservoir for lipophilic antibiotics, which impacts the rate and duration of exposure of intracellular bacteria to these drugs, highlights the importance of in vivo pharmacokinetics in the evaluation and administration of antibiotics to combat human infection.Nanoscale secondary ion mass spectrometry (NanoSIMS) is a spectroscopy approach to interrogate the chemical composition of prokaryotic and eukaryotic cells. Here, a focused ion beam is used to scan the sample surface and sputtered secondary ions are analysed by mass spectrometry34. In both cases, S2 cells (derived from a Drosophila macrophage-like lineage) were treated with a genome-wide siRNA library and subsequently infected with fluorescently labelled Listeria monocytogenes or Mycobacterium fortuitum  to assess intracellular growth. Since these seminal studies, ‘high-throughput’ and ‘high-content screening’ have been accelerated by the emergence of genome-wide ‘CRISPR libraries’, and technological developments associated with automated microscopy which includes advanced acquisition hardware  and improved image processing and analysis tools. Considering the vast amount of data generated, automated pipelines are paramount for fast, unbiased and accurate quantifications. While commercial automated cell imagers are often coupled to dedicated analysis software, many open-access options have become available in the last decade. Some of these perform machine-learning-based classification of phenotypes  or segmentation of precise structures . Recently, machine-learning-based image analysis platforms (e.g. HRMAn38) have been developed for the dedicated analysis of host–pathogen interactions. When applied to Salmonella infection of HeLa cells, the percentage of bacteria decorated with ubiquitin were quantified in an automated manner38. To date, high-throughput and high-content screenings have been performed for different bacterial infections including L. pneumophila, C. trachomatis and M. marinum (reviewed in Brodin and Christophe39). Quantitative studies are focused on bacterial morphology using bacteria in broth culture. In this case, essential genes of B. subtilis were knocked down using CRISPR, and multiple morphological parameters were analysed using an automated single-cell analysis workflow40. The authors categorised a wide range of bacterial cell phenotypes based on their morphology and linked phenotypes to gene networks. It will be important to perform similar studies during infection and determine whether bacteria also undergo similar morphological modifications under antibiotic treatment in vivo.In addition to searching for antibiotic and anti-infectious compounds, screens have provided in depth understanding of the bacterial infection process Fig. . In 2005Escherichia coli)41 and APEX (an ascorbate peroxidase from soybean)42. Proximity biotinylation has been used to investigate the composition and assembly of the T6SS in enteroaggregative E. coli43. Here, APEX2 was tagged to the T6SS key component TssA, which identified TagA, a protein that stops polymerisation of the T6SS sheath and limits its contraction. In addition, the authors expressed APEX2-TssA in various mutants blocked at different stages of T6SS assembly. This approach enabled the temporal dissection of protein interactions required for T6SS biogenesis. Other groups have explored the use of biochemical mapping during infection of tissue culture cells. For example, BioID was used to identify interaction partners of S. Typhimurium T3SS effectors44. Considering that introducing lengthy tags on secreted bacterial effectors frequently impairs their translocation across the bacterial membrane, the authors expressed tagged effectors directly in human epithelial cells. From this, they identified ~400 protein partners for the selected effectors, confirming previously known interactions and also discovering unknown ones, such as the biogenesis of lysosome-related organelle complex (BLOC)-2 as a host target of SifA, an important Salmonella effector required for the formation of Salmonella induced filaments (SIFs) at the BCV. In a study using C. trachomatis, authors used APEX2 to tag two inclusion membrane proteins (IncA and IncF) without impairing their secretion45. Inc proteins are T3SS translocated effectors that incorporate into inclusions bodies, the intracellular vacuole that sustains C. trachomatis replication. Since Inc proteins contain large hydrophobic regions that make their immunoprecipitation challenging, APEX2 tagging was efficient to identify their molecular partners, including LRRF1 (a host protein important for inclusion biogenesis and acquisition of host nutrients by bacteria). It is increasingly recognised that proximity-labelling techniques offer potential to map host–pathogen interactions, considering their success in multiple cell biology studies mapping the proteomic composition of subcellular regions (such as the mitochondria-ER contact sites46). A key advantage is that labelling occurs in live cells, which limits detection of non-specific interactions (e.g. proteins that belong to separate cellular compartments) normally detected by traditional pull downs. In addition, biochemical mapping has been useful to identify transient protein interactions . Recently, labelling enzymes have progressively been modified to achieve higher reaction efficiency and shorter labelling times . This, together with the use of fragment complementation for several enzymes, will ensure a broader application of biochemical mapping in cellular microbiology.Affinity purification followed by mass spectrometry is a traditional approach to identify interaction partners of host or bacterial factors. However, enzyme-catalysed proximity-labelling techniques  can be used as a powerful alternative Fig. . Here, a49. An important advance in this field has been the development of ‘dual RNA-seq’, which enables the simultaneous transcriptome analysis from both host and pathogen, thus capturing the reciprocal response between both organisms in a single experiment49  showed little overlap between their transcripts and differed substantially in the host cell inflammatory response. By integration of their transcriptional profiles with comparative genomics and proteomics, authors identified antisense RNA as a major mechanism of gene regulation in this neglected pathogen. Future dual RNA-seq studies will benefit from ongoing developments in sensitivity offered by third-generation sequencing techniques. In addition, further insights will be gained from the application of this technique to infected tissues (to understand the complexity underlying the infection process of different cell types) and to single-cells52 .A separate method for the exploration of host–pathogen interactions includes the profiling of RNA expression patterns that occur during infection. In a typical transcriptomic experiment, RNA is isolated from the sample of interest, reverse transcribed to cDNA, sometimes amplified, sequenced and mapped to a genome of reference. Understanding RNA expression profiles of bacteria collected from infected samples compared to broth has provided unexpected insights on pathogenic adaptations to infectiont49 Fig. . One sigM. smegmatis to isoniazid, an antitubercular antibiotic53. Due to the disparity in size between bacterial pathogens (~1 μm) and host cells (~50 μm), the design of microfluidic devices for their co-culture has been a challenge. A successful design includes InfectChip, a microfluidic device developed for long-term single-cell time-lapse microscopy of bacterial cell—amoeba interactions, that can be potentially adapted for other host cells, including macrophages54. InfectChip has been used to dissect the diverse outcomes of Klebsiella pneumoniae and M. marinum infection of the soil amoeba Dictyostelium discoideum55. This approach has been shown to effectively monitor and quantify early and late events during host–pathogen interplay at the single-cell level. These events, difficult to analyse using standard microscopy techniques at the bulk population level, were revealed to occur in an asynchronous and heterogeneous manner. In addition, microfluidic devices allow timely control of the perfusion medium, which can be modified during the course of the infection experiment to study the impact of starvation or antibiotics.The incorporation of ‘microfluidic chips’ into microscopy routines has revolutionised the analysis of single bacterial cells and their infection process Fig. . GeneralS. flexneri56. Here, human colonic epithelial cells proliferate and develop into villi- and crypt-like structures on a semipermeable membrane coated with an extracellular matrix. This 3D arrangement of the epithelium was instrumental for T3SS expression and efficient invasion of S. flexneri, which increased ~10,000-fold . In addition, the application of mechanical forces  in Intestine-Chip significantly improved this invasion step. These results challenge a long-standing dogma for S. flexneri invasion: S. flexneri were viewed to invade ‘M cells’ of the intestinal ‘Peyer’s patches’ and subsequently spread basolaterally through the enteric epithelium. Although this dogma could explain the low invasion efficiency of S. flexneri in traditional cell culture systems, these results using Intestine-Chip show that recapitulation of 3D characteristics and mechanical forces of the invaded tissue are essential for the early steps of the infection process. Another microfluidic device, called Lung-on-Chip, has been used to recreate the microenvironment of pulmonary alveoli during infection with M. tuberculosis57. In this microfluidic device, a porous membrane supports the growth of a mixture of endothelial cells found in lung alveoli and GFP expressing bone marrow-derived macrophages. Here, the authors found that defective surfactant production by alveolar epithelial cells resulted in faster intracellular growth of M. tuberculosis. The addition of exogenous surfactant partially removed virulence-associated lipids from the M. tuberculosis cell surface, and impaired their growth in macrophages, highlighting the role of pulmonary surfactant for innate immune defence against tuberculosis. Taken together, organ-on-chip strategies can enable the isolated investigation of key processes underlying infection of complex tissues and organs, difficult to perform in vivo using animal models.Different microfluidic chips have been developed to mimic the environment of infected human organs in a controlled manner Fig. . This isHelicobacter pylori58, S. Typhimurium59 and several pathotypes of E. coli60. In all cases, bacteria are microinjected into the lumen of spherical enteroids, where they invade enterocytes and colonise the epithelium. Injecting bacteria in individual organoids is typically performed manually, and reduces the throughput potential of the experiment. However, recently developed apical-out enteroids revert this polarity, and the apical side of the epithelium is exposed to the external medium. Using this approach, infection with S. Typhimurium occurs upon the addition of bacteria to the medium61. While most studies using enteroids have focused on the integrity of the epithelial barrier during infection, the future of organoids has great potential. Organoids are amenable to several genetic manipulation techniques and represent an exciting platform for drug screens. In addition, human-derived organoids can enable the investigation of commensal and pathogenic bacteria that are restricted to humans . Considering that organoids can be derived from human biopsies that maintain a patient’s genetic background, it is tempting to speculate that organoids will be key for the future development of personalised treatments.Considering their biomedical relevance, ‘organoids’ are promising infection models to study bacterial pathogenesis, as they mimic in vivo characteristics of human organs while still amenable to experimental manipulation Fig. . The typTaken together, chips, organ-on-chips and organoids represent promising platforms to assess complex infection phenotypes in a highly controlled manner, despite some limitations Fig. . Chips a: Caenorhabditis elegans (worm), Drosophila melanogaster (fruit fly), Danio rerio (zebrafish) and Mus musculus (mouse), which have significantly contributed to our understanding of the infection process in vivo 62. Although other bacterial pathogens can infect C. elegans, including S. flexneri, L. monocytogenes and L. pneumophila, only S. Typhimurium can cause intracellular infection . Significantly, C. elegans has been informative to study the role of biofilm formation64 and microbiota65 in vivo. Enterococcus faecium is a human commensal bacterium used as a probiotic in livestock, yet its mechanism of protection against enteric infection was unknown. In a recent study using C. elegans, E. faecium was shown to secrete a peptidoglycan hydrolase that produces peptidoglycan fragments improving host ‘tolerance’ to infection by S. Typhimurium66. This study suggested that enzymes (such as E. faecium peptidoglycan hydrolases) from commensal bacteria can be used to enhance probiotic treatments.Due to its ease of genetic manipulation, rapid generation time, simple body plan and transparency, the nematode D. melanogaster. Infectious disease research using Drosophila is highlighted by the discovery of the innate immune receptor Toll and subsequent identification of homologous Toll-like receptors (TLRs) in humans and mice. TLRs recognise conserved molecules present in microbes, called pathogen-associated molecular patterns (PAMPS), and lead to activation of innate immune signalling pathways. The strength of Drosophila as an infection model is due to its conserved innate immune pathways with mammals, small size and fast generation time, as well as the availability of genetic manipulation tools (making it amenable to screening campaigns). Drosophila has been used to perform genetic screens at the whole organism level to identify factors underlying host resistance to L. monocytogenes infection67. Surprisingly, whole organism results failed to significantly overlap with results from S2 Drosophila derived cells33, showing the importance of doing screens at both the single-cell (studying cell-autonomous immunity) and whole organism level (studying the complex interplay between immune cells and infected tissues)68. Drosophila has also been used to model gastrointestinal infection69 and the role of commensal microbiota in host defence70. Finally, Drosophila is an important model to study the interplay between metabolism and innate immune responses. As one example, cytokines have been shown to dysregulate the autophagy-mediated accumulation of triglycerides in Drosophila macrophages, which promotes M. marinum survival71.A widespread invertebrate model for infection is 72. In addition, their optical transparency is exceptional for non-invasive in vivo microscopy of innate immune development and host–pathogen interactions. The imaging potential of zebrafish larvae has recently been showcased by the development of LLSM in combination with adaptive optics. This provides subcellular imaging with an extraordinary spatiotemporal resolution to visualise multiple biological processes in vivo, including organelle dynamics or neural circuit development73. Infection of zebrafish larvae with M. marinum (a fish pathogen) is recognised as a natural host–pathogen system to model mycobacterial infection in humans. Zebrafish infection is often used to investigate roles for bacterial autophagy in vivo75, and has recently elucidated roles for the pro-inflammatory cytokine tumour necrosis factor (TNF) during necrosis of infected macrophages76. In this case, the intracellular pathway that mediates a TNF response involves multiple interconnected organelles including the mitochondria, lysosome and ER77. Interestingly, this discovery identified several druggable targets with therapeutic potential (such as ER ryanodine receptors). The repertoire of pathogens studied using the zebrafish model has significantly expanded to now include a wide variety of bacterial pathogens, including Mycobacterium leprae, P. aeruginosa and V. cholerae, underscoring their versatility and discovery potential (reviewed in Gomes and Mostowy72). Infection in the hindbrain ventricle has been useful to study intra- and inter-species bacterial interactions, such as competition and predation, inside the host. Signals to induce the Staphylococcus aureus T7SS are poorly understood, yet its activity during zebrafish infection can be exploited to test the function of T7SS effectors in vivo, including TspA shown to depolarise the membrane of competitor bacteria78. In a separate study, predatory bacteria Bdellovibrio bacteriovorus were shown to cooperate with the host innate immune system to clear antimicrobial-resistant S. flexneri, which may inspire the use of predatory bacteria as an alternative to antibiotics to treat bacterial infection in humans79.Zebrafish larvae have transformed our understanding of host–pathogen interactions because of their fully annotated genome (highly homologous to that of humans), genetic manipulability and translational potentialShigella, a neuronal apoptosis inhibitory protein (NAIP)- NLR family CARD domain-containing protein 4 (NLRC4) ‘inflammasome’ deficient mouse model has been generated, which is susceptible to S. flexneri and recapitulates the clinical characteristics of bacillary dysentery in humans80. Importantly, these results suggest that the lack of inflammasome response in human epithelial cells is a major reason for the efficient colonisation of the human intestine by S. flexneri. However, results using mice do not always recapitulate results using tissue culture cells. As a consequence, alternative animal models are being explored. One example is the use of infant rabbits to study S. flexneri infection, which reproduces the bloody diarrhoea phenotype and serves to dissect processes of bacterial invasion and dissemination in vivo81. Another example in which the mouse model did not reflect results obtained in vitro is that of bacterial autophagy, where a study in ATG5 deficient mice showed increased susceptibility to M. tuberculosis infection independent of canonical autophagic activity82.Mice have been the premiere animal model to study infectious diseases for many years, and are necessary for preclinical trials to test the dose and efficacy of treatments and vaccines. Production of knockout mice in inbred strains (including C57BL/6 or BALB/c) are typically used to demonstrate the in vivo relevance of host defence mechanisms discovered previously in tissue culture cells or non-vertebrate animal models. In the case of S. Typhimurium (which causes a typhoid-like disease in mice) have been proposed83. Commensal Bacteroides species have been shown to produce the short fatty acid propionate, which disrupts intracellular pH and prevents S. Typhimurium proliferation84. A major limitation of the mouse model is their natural resistance to some enteropathogenic bacteria, yet humanised mice have been a successful strategy to increase the repertoire of pathogens studied in vivo. For infection by S. enterica serovar Typhi , immunocompromised mice can be grafted with human immune cells to support bacterial proliferation85. In the case of L. monocytogenes infection, a key innovation was the development of transgenic mice expressing human E-cadherin in enterocytes86. Human E-cadherin (which differs from mouse E-cadherin at amino acid position 16) can interact with the L. monocytogenes invasion protein InlA, thus promoting bacterial internalisation. These humanised mice are susceptible to Listeria infection, and were transformative to study bacterial invasion of the intestinal epithelium86 and placental barrier87. Recent studies have shown that species-specific to the mouse microbiota  significantly promote resistance to colonisation by L. monocytogenes88. While targeting specific bacterial competitors  may generate more relevant mouse models to study L. monocytogenes infection, their protective potential when administered as probiotics in humans remains to be studied. Alternatively, transient depletion of mouse microbiota may favour intestinal exposure to engineered L. monocytogenes carrying a highly immunogenic antigen89. This approach to boost the immune response may be a useful platform to study vaccination at the intestinal mucosa in humans.To understand the role of microbiota in gut colonisation by bacterial pathogens, a variety of mouse models for studying infection resistance to Despite their limitations to fully capture the physiopathology of human infections, animal models remain a cornerstone in cellular microbiology to study the complexity of host–pathogen interactions. While only a fraction of human pathogens has been studied using animal models, their increasing application to study unknown bacterial species will accelerate our ability to combat emerging and neglected infectious diseases.Box 2 Modelling bacterial infection at the whole animal levelCaenorhabditis elegans (worm): C. elegans is a transparent unsegmented nematode first established as a model organism by Sidney Brenner to study neural development. The use of C. elegans has enabled fundamental advance significantly contributing to multiple aspects of biology. This is highlighted by the Nobel Prize-winning discovery of short interference RNA (siRNA), which inspired targeted protein depletion and genome-wide screens in a wide range of tissue culture cells and animal models. For infection biology, C. elegans has been used to study antimicrobial peptides and the lack of professional phagocytic cells makes worms convenient to investigate gut epithelial cell immunity. C. elegans is also used for innovative transgenerational studies, investigating mechanisms underlying inheritance of behaviour and pathogen avoidance.Drosophila melanogaster (fruit fly): D. melanogaster is a non-vertebrate model organism first used by Thomas Hunt Morgan to study genetic inheritance. Research using D. melanogaster has been awarded several Nobel Prizes, including one for research on the Toll signalling pathway which motivated the characterisation of Toll-like receptors (TLRs) as sensors of innate immunity in humans and other animals. Since then, D. melanogaster is widely used to study host factors involved in infection (due to its genetic tractability and the wide variety of mutants available) and is mostly focused on antimicrobial peptides, humoral immunity and macrophage-like haemocytes (D. melanogaster primary immune cells). Recent studies are modelling infection in the fly gut epithelium to study the role of the microbiota, diet and metabolism in host defence.Danio rerio (zebrafish): pioneered by George Streisinger to study vertebrate development, D. rerio is an important animal model for biomedical research. The genetic tractability of zebrafish and its amenability to non-invasive high-resolution microscopy makes D. rerio ideal to investigate host–pathogen interactions from the single cell to the whole animal level. Considering that zebrafish larvae lack an adaptive immune response, studies are often performed to understand the role of the innate immune system (in particular macrophages and neutrophils) in host defence. Zebrafish are emerging as a versatile system to study cell-autonomous immunity , bacteria–bacteria interactions  and to discover unforeseen mechanisms of infection control.Mus musculus (mouse): M. musculus is a small rodent and (since its first use in the 17th century by William Harvey) has become the mostly used mammalian model in biomedical research because of its close proximity to human physiology. In vivo research using mice is widely performed due to the large community of inbred and genetically modified transgenic lines available, useful to map or study host factors underlying resistance/susceptibility to infection. Moreover, a variety of ‘humanised’ models have been developed to render mice susceptible to human-specific pathogens. Infection of mice is clinically relevant and routine before advancing clinical trials testing pharmacologic treatments on humans.90, predicting molecules with an antibacterial activity using machine learning91 and/or promoting treatments to precisely modulate the patient’s immune response. Regarding antimicrobials, more studies are needed to address the impact of clinically-approved drugs on the microbiota to provide a more tailored administration that benefits the individual patient. While Listeria, Shigella and Salmonella are recognised as paradigms for cellular microbiology research, increasing the repertoire of bacterial pathogens under investigation will be important to discover unexpected pathogenic mechanisms required for host cell infection. The development of tools for the investigation of genetically intractable organisms, including the obligate intracellular bacteria Rickettsia92 and Orientia51, is rendering poorly understood pathogens more amenable to cellular microbiology research. Considering the adaptation of bacterial strains to the lab, increasing the study of clinical isolates for cellular microbiology will be invaluable to capture the full breadth of pathogenic agents and mechanisms of disease circulating in the human population. In terms of the host, the study of human genetic immunodeficiencies that predispose to infection (i.e. the human model) in combination with cellular microbiology has great potential to directly impact public health. As one example, inborn genetic errors in type-I-IFN immunity has been shown to predispose to severe COVID-19 disease94. Finally, increasing the technical capacity and throughput of manipulating patient cells and tissues in vitro can help to address the heterogeneity underlying human susceptibility to infection, and promote the exploration of personalised treatments.A more complete understanding of host–pathogen interactions is required to improve therapeutics for human health. Here, we overview innovative technologies transforming the field of cellular microbiology, enabling fundamental discovery in both infection and cell biology. However, the field faces important challenges to influence global health in an efficient manner  and animal models have transformed approaches to study cellular microbiology. For an improved understanding of pathogenesis and fundamental cellular processes, how faithfully do these platforms/animal models have to model natural human infection?We are only beginning to understand bacteria–bacteria interactions and the role of complex bacterial secretion systems. In this context, what is the influence of infection (and subsequent antibiotic treatment) on microbiota, community architecture and whole animal health?Listeria, Shigella and Salmonella are paradigms of cellular microbiology research. How can the investigation of poorly understood bacterial species and clinical strains expand our understanding of the infected cell?For over 30 years, cellular microbiology has provided extensive insights on mechanisms of bacterial pathogenesis and host response. How will advances in the field shape future medicine and more broadly impact human health?