Living Host Cells Research Articles

AGC family kinase of Entamoeba histolytica: Decoding the members biochemically.

Entamoeba histolytica, a protozoan parasite, is the causative agent of amoebiasis, which is a significant global health concern. The virulence mechanisms underlying its pathogenicity are multifaceted and complex. However, endocytic processes and motility are well accepted virulence determinants. As previously reported, an AGCK family kinase, EhAGCK1 to be involved in trogocytosis exclusively while another one from same family named EhAGCK2 participates in all actin dependent endocytic processes. As the kinase dead mutants of EhAGCK1 showed significant defect in destruction of live host cells and also the localisation pattern of same is distinguishable from EhAGCK2. From observations so far, it appears that former initiates a distinguishable signaling cascade. In this work, we have demonstrated distinct biochemical properties of kinases involved in related yet distinguishable endocytic processes for the first time. Our biochemical characterization highlights distinct ion dependency of EhAGCK1 along with substrate specificity. We also show upstream activator of these kinases, 3-phosphoinositide dependent kinase 1 (PDK1) activity and its role in activating the kinase activity. The kinases exhibit property of autophosphorylation, and which may regulate the kinase activity subsequently. Summarily, these studies show that EhAGCK1 and EhAGCK2 show distinct biochemical properties which further confirm their unique role in related endocytic processes of trogocytosis and phagocytosis.

Direct Visualization of Hepatitis C Virus RNA in Living Cells and Mice.

RNA virus infection is a global health issue with a significant economic burden. Direct visualization of the viral RNA genome in living cells is crucial for virological research and early clinical diagnosis. Thus, the need to continue research to find imaging toolkits is urgent. The RNA G-quadruplex (G4), a noncanonical secondary structure with stacked planar G-quartets, has recently been demonstrated in the RNA genomes of various viruses, especially the hepatitis C virus (HCV). Recent advancements in small molecular fluorescent probes have paved the way for a novel method of visualizing RNA G4s. Herein, we describe a fluorogenic probe-based RNA G4s light-up system for visualizing the HCV genome in living host cells and HCV RNA-presenting mini-organ-bearing mice without complicated sample pretreatment. Using this approach, we achieved (i) the visualization of HCV RNA genome in living cells, (ii) the investigation of viral RNA subcellular distribution, (iii) the dynamic tracking of native HCV infection and propagation in the host cell, and (iv) the high-contrast HCV RNA imaging in living mice bearing the HCV RNA-presenting mini-organ.

Experimental uncoupling of hosts and endosymbionts.

Many organisms harbor heritable bacterial symbionts that offer context-specific benefits to their hosts. In some of these symbioses, symbionts live inside host cells as endosymbionts. Studying the biology of endosymbiosis is challenging because it is hard to independently cultivate hosts and endosymbionts. A recent study, using a simple defined growth medium at ambient temperature, established an axenic culture of the pea aphid's heritable bacterial endosymbiont, Candidatus Fukatsuia symbiotica (G. P. Maeda, M. K. Kelly, A. Sundar, and N. A. Moran, mBio 15:e03253-23, 2024, https://doi.org/10.1128/mbio.03253-23). Notably, the monoculture was capable of host recolonization, was stably transmitted, and returned similar host phenotypes to those observed in native infections. This advance in uncoupling the cultivation of an endosymbiont and its host opens avenues for genetic manipulation of the endosymbiont that will facilitate hypothesis-driven work to explore the mechanisms of host-endosymbiont biology and potentially facilitate the development of symbiont-mediated practical-application biotechnologies.

Molecular Engineering of Functional SiRNA Agents.

Synthetic biology constitutes a scientific domain focused on intentional redesign of organisms to confer novel functionalities or create new products through strategic engineering of their genetic makeup. Leveraging the inherent capabilities of nature, one may address challenges across diverse sectors including medicine. Inspired by this concept, we have developed an innovative bioengineering platform, enabling high-yield and large-scale production of biological small interfering RNA (BioRNA/siRNA) agents via bacterial fermentation. Herein, we show that with the use of a new tRNA fused pre-miRNA carrier, we can produce various forms of BioRNA/siRNA agents within living host cells. We report a high-level overexpression of nine target BioRNA/siRNA molecules at 100% success rate, yielding 3-10 mg of BioRNA/siRNA per 0.25 L of bacterial culture with high purity (>98%) and low endotoxin (<5 EU/μg RNA). Furthermore, we demonstrate that three representative BioRNA/siRNAs against GFP, BCL2, and PD-L1 are biologically active and can specifically and efficiently silence their respective targets with the potential to effectively produce downstream antiproliferation effects by PD-L1-siRNA. With these promising results, we aim to advance the field of synthetic biology by offering a novel platform to bioengineer functional siRNA agents for research and drug development.

Quantitative analysis of morphogenesis and growth dynamics in an obligate intracellular bacterium.

Obligate intracellular bacteria of the order Rickettsiales include important human pathogens. However, our understanding of the biology of Rickettsia species is limited by challenges imposed by their obligate intracellular lifestyle. To overcome this roadblock, we developed methods to assess cell wall composition, growth, and morphology of Rickettsia parkeri, a human pathogen in the spotted fever group of the Rickettsia genus. Analysis of the cell wall of R. parkeri revealed unique features that distinguish it from free-living alphaproteobacteria. Using a novel fluorescence microscopy approach, we quantified R. parkeri morphology in live host cells and found that the fraction of the population undergoing cell division decreased over the course of infection. We further demonstrated the feasibility of localizing fluorescence fusions, for example, to the cell division protein ZapA, in live R. parkeri for the first time. To evaluate population growth kinetics, we developed an imaging-based assay that improves on the throughput and resolution of other methods. Finally, we applied these tools to quantitatively demonstrate that the actin homologue MreB is required for R. parkeri growth and rod shape. Collectively, a toolkit was developed of high-throughput, quantitative tools to understand growth and morphogenesis of R. parkeri that is translatable to other obligate intracellular bacteria.

Single molecule analyses reveal dynamics of Salmonella translocated effector proteins in host cell endomembranes

The facultative intracellular pathogen Salmonella enterica remodels the host endosomal system for survival and proliferation inside host cells. Salmonella resides within the Salmonella-containing vacuole (SCV) and by Salmonella-induced fusions of host endomembranes, the SCV is connected with extensive tubular structures termed Salmonella-induced filaments (SIF). The intracellular lifestyle of Salmonella critically depends on effector proteins translocated into host cells. A subset of effectors is associated with, or integral in SCV and SIF membranes. How effectors reach their subcellular destination, and how they interact with endomembranes remodeled by Salmonella remains to be determined. We deployed self-labeling enzyme tags to label translocated effectors in living host cells, and analyzed their single molecule dynamics. Translocated effectors diffuse in membranes of SIF with mobility comparable to membrane-integral host proteins in endomembranes. Dynamics differ between various effectors investigated and is dependent on membrane architecture of SIF. In the early infection, host endosomal vesicles are associated with Salmonella effectors. Effector-positive vesicles continuously fuse with SCV and SIF membranes, providing a route of effector delivery by translocation, interaction with endosomal vesicles, and ultimately fusion with the continuum of SCV/SIF membranes. This mechanism controls membrane deformation and vesicular fusion to generate the specific intracellular niche for bacterial survival and proliferation.

SARS-CoV-2 SUD2 and Nsp5 Conspire to Boost Apoptosis of Respiratory Epithelial Cells via an Augmented Interaction with the G-Quadruplex of BclII.

The molecular mechanisms underlying how SUD2 recruits other proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to exert its G-quadruplex (G4)-dependent pathogenic function is unknown. Herein, Nsp5 was singled out as a binding partner of the SUD2-N+M domains (SUD2core) with high affinity, through the surface located crossing these two domains. Biochemical and fluorescent assays demonstrated that this complex also formed in the nucleus of living host cells. Moreover, the SUD2core-Nsp5 complex displayed significantly enhanced selective binding affinity for the G4 structure in the BclII promoter than did SUD2core alone. This increased stability exhibited by the tertiary complex was rationalized by AlphaFold2 and molecular dynamics analysis. In line with these molecular interactions, downregulation of BclII and subsequent augmented apoptosis of respiratory cells were both observed. These results provide novel information and a new avenue to explore therapeutic strategies targeting SARS-CoV-2. IMPORTANCE SUD2, a unique protein domain closely related to the pathogenesis of SARS-CoV-2, has been reported to bind with the G-quadruplex (G4), a special noncanonical DNA structure endowed with important functions in regulating gene expression. However, the interacting partner of SUD2, among other SARS-CoV-2 Nsps, and the resulting functional consequences remain unknown. Here, a stable complex formed between SUD2 and Nsp5 was fully characterized both in vitro and in host cells. Moreover, this complex had a significantly enhanced binding affinity specifically targeting the Bcl2G4 in the promoter region of the antiapoptotic gene BclII, compared with SUD2 alone. In respiratory epithelial cells, the SUD2-Nsp5 complex promoted BclII-mediated apoptosis in a G4-dependent manner. These results reveal fresh information about matched multicomponent interactions, which can be parlayed to develop new therapeutics for future relevant viral disease.

Recombinant Reporter Phage rTUN1::nLuc Enables Rapid Detection and Real-Time Antibiotic Susceptibility Testing of Klebsiella pneumoniae K64 Strains.

The emergence of multi-drug-resistant Klebsiella pneumoniae (Kp) strains constitutes an enormous threat to global health as multi-drug resistance-associated treatment failure causes high mortality rates in nosocomial infections. Rapid pathogen detection and antibiotic resistance screening are therefore crucial for successful therapy and thus patient survival. Reporter phage-based diagnostics offer a way to speed up pathogen identification and resistance testing as integration of reporter genes into highly specific phages allows real-time detection of phage replication and thus living host cells. Kp-specific phages use the host's capsule, a major virulence factor of Kp, as a receptor for adsorption. To date, 80 different Kp capsule types (K-serotypes) have been described with predominant capsule types varying between different countries and continents. Therefore, reporter phages need to be customized according to the locally prevailing variants. Recently, we described the autographivirus vB_KpP_TUN1 (TUN1), which specifically infects Kp K64 strains, the most predominant capsule type at the military hospital in Tunis (MHT) that is also associated with high mortality rates. In this work, we developed the highly specific recombinant reporter phage rTUN1::nLuc, which produces nanoluciferase (nLuc) upon host infection and thus enables rapid detection of Kp K64 cells in clinical matrices such as blood and urine. At the same time, rTUN1::nLuc allows for rapid antibiotic susceptibility testing and therefore identification of suitable antibiotic treatment in less than 3 h.

Botrytis hypersensitive response inducing protein 1 triggers noncanonical PTI to induce plant cell death.

According to their lifestyle, plant pathogens are divided into biotrophic and necrotrophic organisms. Biotrophic pathogens exclusively nourish living host cells, whereas necrotrophic pathogens rapidly kill host cells and nourish cell walls and cell contents. To this end, the necrotrophic fungus Botrytis cinerea secretes large amounts of phytotoxic proteins and cell wall-degrading enzymes. However, the precise role of these proteins during infection is unknown. Here, we report on the identification and characterization of the previously unknown toxic protein hypersensitive response-inducing protein 1 (Hip1), which induces plant cell death. We found the adoption of a structurally conserved folded Alternaria alternata Alt a 1 protein structure to be a prerequisite for Hip1 to exert its necrosis-inducing activity in a host-specific manner. Localization and the induction of typical plant defense responses by Hip1 indicate recognition as a pathogen-associated molecular pattern at the plant plasma membrane. In contrast to other secreted toxic Botrytis proteins, the activity of Hip1 does not depend on the presence of the receptor-associated kinases BRI1-associated kinase 1 and suppressor of BIR1-1. Our results demonstrate that recognition of Hip1, even in the absence of obvious enzymatic or pore-forming activity, induces strong plant defense reactions eventually leading to plant cell death. Botrytis hip1 overexpression strains generated by CRISPR/Cas9 displayed enhanced infection, indicating the virulence-promoting potential of Hip1. Taken together, Hip1 induces a noncanonical defense response which might be a common feature of structurally conserved fungal proteins from the Alt a 1 family.

Visualizing Influenza A Virus vRNA Replication.

Influenza A virus (IAV) has caused recurrent epidemics and severe pandemics. In this study, we adapted an MS2-MCP live-cell imaging system to visualize IAV replication. A reporter plasmid, pHH-PB2-vMSL, was constructed by replacing a part of the PB2-coding sequence in pHH-PB2 with a sequence encoding 24 copies of a stem-loop structure from bacteriophage MS2 (MSL). Binding of MS2 coat protein (MCP) fused to green fluorescent protein (GFP) to MSL enabled the detection of vRNA as fluorescent punctate signals in live-cell imaging. The introduction of pHH-PB2-vMSL into A549 cells transduced to express an MCP-GFP fusion protein lacking the nuclear localization signal (MCP-GFPdN), subsequently allowed tracking of the distribution and replication of PB2-vMSL vRNA after IAV PR8 infection. Spatial and temporal measurements revealed exponential increases in vRNA punctate signal intensity, which was only observed after membrane blebbing in apoptotic cells. Similar signal intensity increases in apoptotic cells were also observed after MDCK cells, transduced to express MCP-GFPdN, were infected with IAV carrying PB2-vMSL vRNA. Notably, PB2-vMSL vRNA replication was observed to occur only in apoptotic cells, at a consistent time after apoptosis initiation. There was a lack of observable PB2-vMSL vRNA replication in non-apoptotic cells, and vRNA replication was suppressed in the presence of apoptosis inhibitors. These findings point to an important role for apoptosis in IAV vRNA replication. The utility of the MS2-imaging system for visualizing time-sensitive processes such as viral replication in live host cells is also demonstrated in this study.

Live imaging of Yersinia translocon formation and immune recognition in host cells.

Yersinia enterocolitica employs a type three secretion system (T3SS) to translocate immunosuppressive effector proteins into host cells. To this end, the T3SS assembles a translocon/pore complex composed of the translocator proteins YopB and YopD in host cell membranes serving as an entry port for the effectors. The translocon is formed in a Yersinia-containing pre-phagosomal compartment that is connected to the extracellular space. As the phagosome matures, the translocon and the membrane damage it causes are recognized by the cell-autonomous immune system. We infected cells in the presence of fluorophore-labeled ALFA-tag-binding nanobodies with a Y. enterocolitica strain expressing YopD labeled with an ALFA-tag. Thereby we could record the integration of YopD into translocons and its intracellular fate in living host cells. YopD was integrated into translocons around 2 min after uptake of the bacteria into a phosphatidylinositol-4,5-bisphosphate enriched pre-phagosomal compartment and remained there for 27 min on average. Damaging of the phagosomal membrane as visualized with recruitment of GFP-tagged galectin-3 occurred in the mean around 14 min after translocon formation. Shortly after recruitment of galectin-3, guanylate-binding protein 1 (GBP-1) was recruited to phagosomes, which was accompanied by a decrease in the signal intensity of translocons, suggesting their degradation or disassembly. In sum, we were able for the first time to film the spatiotemporal dynamics of Yersinia T3SS translocon formation and degradation and its sensing by components of the cell-autonomous immune system.

Articles of Significant Interest in This Issue

Birkle and Brown (e00476-20) propose and review evidence for a novel mode of innate immunity whereby live, infected host cells induce phagocytes to phagocytose the infected cell, thereby potentially reducing infection. They discuss evidence that live host cells, infected by virus, bacteria, or other intracellular pathogens, release signals that recruit phagocytes to phagocytose the infected cell. Digestion of the infected cell within the phagocyte can curtail host infection but may also result in cross-presentation of pathogen antigens, resulting in innate and adaptive immunity.

Milestone of World Pandemics: A Review on Remedy for COVID-19 Diseases to Revitalize Human Race from Deadly Corona Virus

Viruses are microscopic organisms that can only grow within living host cells. Various types of animals such as Rats, Bat, Cows, snakes, and related wild animals can serve as vehicles of transmission of this group of organisms. Novel SARS-CoV-2 belongs to the Coronaviridae family that was reported to have emanated from Wuhan, China in December 2019. It alerts world response against strange viral respiratory disease. As the world reflects on pandemics historically. Epidemics like the London plague of 1665 and the Plague that emanates from Marseille, France of 1720 claimed millions of lives. Among the most dreadful world pandemics are the “Antonine Plague” which occurred in 165AD and claimed 5 million lives in Egypt, Asia, Italy, and Greece. “Plague of Justinian” was reported to have occurred between the years 541 and 542. In addition to this, “The Flu Pandemic” is also referred to as Spanish flu ravaged the world during the year 1918 to 1920, which is about a century ago.”3rd Plague of 1855” originated from China and was responsible for the death of 10 million Indians within one year.” The Black Death” which is a 7 years long pandemic devastated the world between the years 1346 to 1353. It leads to the death of half of the global population and was by far the worst pandemic. HIV-AIDS has peak record deaths between 2005-2012. In the year 2019/2020 COVID-19 broke out severely than MERS-CoV and SARS-CoV which occurred in recent years. One possible best approach for the control of COVID-19 diseases caused by SARS-CoV-2 is to improve the hosts’ immune system. Evidence shows that inflammatory conditions militate against the immune systems of COVID-19 patients. Some other control strategies will include the use of face masks, physical distancing, hand washing, or use of alcohol-based sanitizers and related personal hygiene are important to contain the diseases.

CoGRIM19 is required for invasive hyphal growth of Colletotrichum orbiculare inside epidermal cells of cucumber cotyledons

Colletotrichum orbiculare, an anthracnose disease fungus of cucurbit plants, extends penetration hyphae inside the epidermal cells of host plants. Unlike vegetative hyphae formed on a nutrient rich medium, this pathogen initially develops biotrophic penetration hyphae, which acquire nutrient resources from living host cells and secret effector proteins to suppress host defense responses. Subsequently, the nature of penetration hyphae changes from biotrophy to necrotrophy in response to the interaction with a host plant. Hence, controlling the extension of penetration hyphae is crucial for C. orbiculare infection. Here, we identified CoGRIM19 encoding Nadh-ubiquinone oxidoreductase subunit as a pathogenicity gene. Pathogenicity assays showed that the cogrim19 mutant caused no visible symptoms on cucumber cotyledons. Microscopic observations revealed that the cogrim19 mutant developed an appressorium and penetration hyphae under artificial conditions such as on coverslips or cellulose membranes, but the penetration hyphae of the mutant were retarded in the cucumber cotyledons. Microscopic observations of biotrophy-specific expression fluorescent signals revealed that the biotrophic stage was maintained in the retarded penetration hyphae of the cogrim19 mutant as the penetration of the wild type. In addition to cytological observations, pathogenicity assays using wounded leaves showed that the cogrim19 mutant had an attenuated pathogenesis. Taking our results together, CoGRIM19 is required for invasive hyphal growth inside the epidermal cells of cucumber cotyledons in C. orbiculare.

Virology and Biopolitics

As neither alive nor dead —though deadly, as we had to learn again—the virus is the figure of the border. The border divides, of course, but thereby relates what it separates. The virus belongs to the living, but on its border, disrupting life from the outside. If Derrida once described deconstruction as a 'virology', it was to note its special interest in the constitutive failure of coding and de-coding, in the interruption of goal-directed reading processes: the virus as parasite that derails communication, whether biological, linguistic, or information-technological (Derrida 1994, 12). Deconstruction seeks to grasp this derailment as necessary for reproduction in general, including understanding as a form of recapitulation. As Derrida also points out in the same context, viruses can become a privileged figure here for another reason: as neither alive nor dead, they effect this disruption from a site that is not itself capable of self-reproduction, a capacity often seen as the very definition of life. A virus is said to be non-living because it can multiply or reproduce only inside living host cells that it hijacks. It has no cellular structure and lacks its own metabolism. But it is not exactly dead either, as it contains genetic information that, alas, can be reproduced.

High-Throughput Sequencing for Deciphering the Virome of Alfalfa (Medicago sativa L.).

Alfalfa (Medicago sativa L.), also known as lucerne, is a major forage crop worldwide. In the United States, it has recently become the third most valuable field crop, with an estimated value of over $9.3 billion. Alfalfa is naturally infected by many different pathogens, including viruses, obligate parasites that reproduce only inside living host cells. Traditionally, viral infections of alfalfa have been considered by breeders, growers, producers and researchers to be diseases of limited importance, although they are widespread in all major cultivation areas. However, over the past few years, due to the rapid development of high-throughput sequencing (HTS), viral metagenomics, bioinformatics tools for interpreting massive amounts of HTS data and the increasing accessibility of public data repositories for transcriptomic discoveries, several emerging viruses of alfalfa with the potential to cause serious yield losses have been described. They include alfalfa leaf curl virus (family Geminiviridae), alfalfa dwarf virus (family Rhabdoviridae), alfalfa enamovirus 1 (family Luteoviridae), alfalfa virus S (family Alphaflexiviridae) and others. These discoveries have called into question the assumed low economic impact of viral diseases in alfalfa and further suggested their possible contribution to the severity of complex infections involving multiple pathogens. In this review, we will focus on viruses of alfalfa recently described in different laboratories on the basis of the above research methodologies.

Cell-Free Synthesis of Natural Compounds from Genomic DNA of Biosynthetic Gene Clusters.

A variety of chemicals can be produced in a living host cell via optimized and engineered biosynthetic pathways. Despite the successes, pathway engineering remains demanding because of the lack of specific functions or substrates in the host cell, the cell's sensitivity in vital physiological processes to the heterologous components, or constrained mass transfer across the membrane. In this study, we show that complex multidomain proteins involved in natural compound biosynthesis can be produced from encoding DNA in vitro in a minimal complex PURE system to directly run multistep reactions. Specifically, we synthesize indigoidine and rhabdopeptides with the in vitro produced multidomain nonribosomal peptide synthetases BpsA and KJ12ABC from the organisms Streptomyces lavendulae and Xenorhabdus KJ12.1, respectively. These in vitro produced proteins are analyzed in yield, post-translational modification and in their ability to synthesize the natural compounds, and compared to recombinantly produced proteins. Our study highlights cell-free PURE system as suitable setting for the characterization of biosynthetic gene clusters that can potentially be harnessed for the rapid engineering of biosynthetic pathways.

Life style of fungi from Biotrophy to Necrotrophy and Saprotrophy

Plant pathogenic fungi causes’ economic menace to crop production throughout the world. On the basis of their life styles they may be classified as biotrophs, hemibiotrophs and necrotrophs. For biotrophs it is mandatory to thrive on living host cell and tissues and often found to secrete a little amount of cell wall degrading enzymes and certain effector molecules for suppressing plant host defense mechanism. Necrotrophs survive on dead host cell and tissues which are killed by them before or during infection. Hemibiotrophs in their early stage of life behave as biotrophs and become necrotrophs on later. This article represents the evolution of biotrophs, interaction of biotrophs, hemibiotrophs and necrotrophs with their host plant and continuum of life styles from biotrophy, through to necrotrophy and ultimately to saprotrophy.

De novo indol-3-ylmethyl glucosinolate biosynthesis, and not long-distance transport, contributes to defence of Arabidopsis against powdery mildew.

Powdery mildew is a fungal disease that affects a wide range of plants and reduces crop yield worldwide. As obligate biotrophs, powdery mildew fungi manipulate living host cells to suppress defence responses and to obtain nutrients. Members of the plant order Brassicales produce indole glucosinolates that effectively protect them from attack by non-adapted fungi. Indol-3-ylmethyl glucosinolate is constitutively produced in the phloem and transported to epidermal cells for storage. Upon attack, indol-3-ylmethyl glucosinolate is activated by CYP81F2 to provide broad-spectrum defence against fungi. How de novo biosynthesis and transport contribute to defence of powdery mildew-attacked epidermal cells is unknown. Bioassays and glucosinolate analysis demonstrate that GTR glucosinolate transporters are not involved in antifungal defence. Using quantitative live-cell imaging of fluorophore-tagged markers, we show that accumulation of the glucosinolate biosynthetic enzymes CYP83B1 and SUR1 is induced in epidermal cells attacked by the non-adapted barley powdery mildew Blumeria graminis f.sp. hordei. By contrast, glucosinolate biosynthesis is attenuated during interaction with the virulent powdery mildew Golovinomyces orontii. Interestingly, SUR1 induction is delayed during the Golovinomyces orontii interaction. We conclude that epidermal de novo synthesis of indol-3-ylmethyl glucosinolate contributes to CYP81F2-mediated broad-spectrum antifungal resistance and that adapted powdery mildews may target this process.

Detection of methicillin-resistant Staphylococcus aureus persistence in osteoblasts using imaging flow cytometry.

Methicillin‐resistant S. aureus has been reported as the main pathogen involved in chronic infections, osteomyelitis, and prosthetic joint infections. The host/pathogen interaction is dynamic and requires several changes to promote bacterial survival. Here, we focused on the internalization and persistence behavior of well‐characterized Staphylococcus aureus invasive strains belonging to the main ST‐MRSA‐SCCmec clones. To overcome the limitations of the cell culture method, we comparatively analyzed the ability of internalization within human MG‐63 osteoblasts with imaging flow cytometry (IFC). After evaluation by cell culture assay, the MRSA clones in the study were all able to readily internalize at 3h postinfection, the persistence of intracellular bacteria was evaluated at 24h both by routine cell culture and IFC assay, after vancomycin‐BODIPY staining. A statistical difference of persistence was found in ST5‐SCCmecII (26.59%), ST228‐SCCmecI (20.25%), ST8‐SCCmecIV (19.52%), ST239‐SCCmecIII (47.82%), and ST22‐SCCmecIVh (50.55%) showing the same ability to internalize as ATCC12598 (51%), the invasive isolate used as control strain for invasion and persistence assays. We demonstrated that the intracellular persistence process depends on the total number of infected cells. Comparing our data obtained by IFC with those of the cell culture assay, we obtained greater reproducibility rates and a number of intracellular bacteria, with the advantage of analyzing live host cells. Moreover, with some limitations related to the lack of whole‐genome sequencing analysis, we validated the different proclivities to persist in the main Italian HA‐MRSA invasive isolates and our results highlighted the heterogeneity of the different clones to persist during cell infection.