Bacteria and Intracellularity

Shigella spp. are diarrheal pathogens closely related to Escherichia coli. They are named after Kiyoshi Shiga, who in 1898 identified its most virulent member, Shigella dysenteriae, as the causative agent of bacillary dysentery, also known as shigellosis ( 1 ). Shigella spp. are Gram-negative, non-spore-forming, facultative anaerobic bacilli that in humans and other primates cause diarrheal disease by invading the colonic epithelium. Spreading of the infection is generally limited to the intestinal lining, where it leads to colonic inflammation, mucosal ulceration, and a loss in intestinal barrier function. Shigellae are transmitted through the fecal-oral route or through ingestion of contaminated food and water. In most cases, Shigella spp. cause a self-limiting disease that can be effectively treated by oral rehydration or antibiotics, although a steady increase in the number of shigellosis cases caused by antibiotic-resistant Shigella strains has become a growing concern ( 2 , 3 ). Shigellosis can be fatal in the very young and in infected individuals who are immunocompromised or do not have access to adequate medical treatment.

Bacterial pathogens typically target specific host tissues. The interaction between host and pathogen is a complex process that differs from cell to cell ( 1 ). It strongly depends on the targeted organ and the pathogen itself. Organs are composed of multiple cell types that may cooperate in antimicrobial defense. While earlier work focused on the role of immune cells, it is becoming increasingly clear that non-hematopoietic cells can also serve as key orchestrators of defense. Pathogens employ a combination of virulence factors in order to ensure nutrient supply, avoid killing by innate defenses, and actively manipulate the host in order to establish infection and fuel transmission ( 2 ). Deciphering virulence factor function during actual infection of an animal host therefore holds the key to understanding the infection process.

With the current understanding that microbes play a large role in shaping human physiology, there has been renewed interest in the study of host-microbe interactions. Much of our present knowledge of host-pathogen interactions has resulted from studies with animal models or cultured immortalized cell lines that attempt to reproduce specific events during host-microbe interactions ( 1 ). Underlying these strategies is the goal of reproducing the important events that occur during human disease. The closest approximation to human infection has been animal infections using model organisms such as the mouse, to attempt to generate a system with complexity and biological functions similar to those of humans. Unfortunately, many important pathogens do not readily colonize or establish disease in common mammalian models, and the response to infection does not always recapitulate the human response ( 2 , 3 ). A particularly confounding problem is that the infection site is often inaccessible in the animal, interfering with the ability to perform manipulations in real time or to make direct observations at the microscopic level.

The endothelium is the layer of endothelial cells lining the inner surface of blood vessels, which span the entire body and ensure the distribution of blood throughout the organism ( 1 ). It is estimated that the human body contains a staggering 100,000 km of blood vessels, more than twice the earth’s circumference ( 2 ). Therefore, a bacterium reaching the circulation is engaged in a network of huge proportions. Moreover, a pathogen traveling through the circulatory system does not encounter a homogeneous environment, as an important feature of the vascular network is its diversity. Although endothelial cells are present in all vessels, the organization of the vessel wall, which is formed by three layers—the tunica intima, media, and adventitia (from the vessel lumen outward)—is different among different vessel types and different organs ( 3 ). Vessels can be first differentiated by the complex extracellular matrix layers surrounding them. For instance, elastic arteries such as the aorta are surrounded by 50 elastic layers, providing them with unique mechanical properties ( 3 ). Second, the cellular content is also different according to vessel type; the walls of arteries and veins contain a layer of smooth muscle cells that gives them the capacity to relax or constrict in response to vasoactive molecules ( 4 ). An additional level of complexity in the network stems from the fact that larger vessels, veins or arteries, are themselves vascularized by smaller vessels, the vasa vasorum ( 5 ).

Urinary tract infections (UTIs) refer to bacterial colonization of the urinary tract and are one of the most common bacterial infections, infecting an estimated 150 million people worldwide annually. In the United States alone, nearly 11 million cases are reported each year, resulting in approximately $5 billion in indirect and direct costs annually ( 1 , 2 ). More than 50% of women will experience at least one UTI in their lifetime, and, despite antibiotic intervention, 20 to 30% of women with an initial UTI will experience a recurrent UTI (rUTI) within 3 to 4 months of the initial infection ( 2 , 3 ). Such infections therefore represent a great health care burden and, as such, demand further research to advance treatment options and improve patient care. This article outlines what is currently known about the determinants and features of Escherichia coli pathogenesis in UTIs and highlights how such knowledge is now being translated into tools for alleviating that burden clinically.

Bacteria of the genus Brucella belong to the alpha-2 subgroup of Alphaproteobacteria, a phylogenetic subgroup which includes a variety of bacteria that are either animal or plant pathogens or symbionts. As such, these bacteria have experienced a long-standing coevolution with eukaryotic hosts that has likely shaped their biology. The genus Brucella is composed of an increasing number of species that infect a wide variety of mammals as primary hosts, such as bovines (Brucella abortus), goats (B. melitensis), swine (B. suis), ovines (B. ovis), camels, elk, bison (B. abortus), canines (B. canis), rodents (B. neotomae and B. microti), and monkeys (B. papionis), as well as marine mammals such as seals, porpoises, dolphins, and whales (B. pinnipedialis and B. ceti) and also amphibians (B. inopinata) ( 1 ). Most species cause in their hosts a disease named brucellosis, which manifests as abortion, sterility, and lameness in animals and which can also be transmitted to humans via inhalation of aerosolized bacteria or via ingestion of, or contact with, contaminated tissues or derived products, classically by the most pathogenic species, B. melitensis, B. suis, and B. abortus, with additional cases due to B. canis and B. neotomae ( 2 – 4 ). Human brucellosis is characterized by nonspecific flu-like symptoms during an early acute phase, which is followed by a chronic infection with debilitating consequences, including recurrent fever, osteomyelitis, arthritis, neurological symptoms, and endocarditis, if not treated with antibiotic therapy in a timely manner ( 4 ). Animal and human brucellosis share common pathophysiological features at the cellular level, where bacteria undergo an intracellular cycle that ensures their survival, proliferation, and persistence within phagocytic cells of various tissues, including macrophages and dendritic cells ( 4 , 5 ). Initially described in placental tissues of infected animals ( 6 ), the ability of B. abortus to extensively proliferate in mammalian cells was reproduced in a variety of tissue culture models of epithelial and phagocytic cells that have been instrumental in defining the main features of the bacterium’s intracellular cycle ( 7 – 12 ).

Tuberculosis (TB) is a global health problem caused by the airborne pathogen Mycobacterium tuberculosis. Currently, one-third of the world’s population is infected with M. tuberculosis, and this slow, tenacious bacterium kills 1.6 million people around the world each year, equating to over 4,300 deaths every day ( 1 ). Failure to eradicate this age-old disease is the result of an ineffective vaccine and extended, often insufficient, chemotherapy. To date, the only licensed vaccine available is Mycobacterium bovis BCG, a live attenuated strain of M. bovis discovered in 1919 by Albert Calmette and Camille Guérin following 230 subcultures of the original virulent isolate ( 2 , 3 ). Distribution of this vaccine to various countries, and more subculturing, led to genetic variations between different BCG strains. However, all strains possess a common deletion that occurred prior to 1919. The deleted region is called region of difference 1 (RD1), and it encodes a key part of the type VII secretion system known as ESAT-6 secretion system 1 (ESX-1) ( Fig. 1A ); deletion(s) in this particular region are considered the major cause of BCG attenuation ( 4 – 6 ).

The foundations of our understanding of intracellular parasitism by a range of eukaryote and prokaryote pathogens has been laid by using tissue culture infection models. These models, using defined cell lines or expanded primary cell cultures, have been invaluable in the generation of the knowledge base on which the field currently relies. However, the models artificially compress the heterogeneity that exists for all these pathogens in their natural in vivo infection cycle. It is the heterogeneity within the pathogen population that enhances a pathogen’s capacity to adapt and survive under the different immune pressures and tissue environments within its host ( 1 – 3 ).

The species of the genus Wolbachia are Gram-negative members of the Alphaproteobacteria that belong to the order Rickettsiales. First discovered in the germ line of mosquitos almost a century ago, they have been shown to have extraordinary diversity, as well as an impressive number of host species ( 1 ). Early on, these endosymbionts received attention from ecologists for the diversity of phenotypes they induce in their terrestrial arthropod hosts. Later, cell and developmental biologists worked to decipher the mechanisms underlying Wolbachia-host interactions, followed by a growing number of experts in all biological disciplines, stimulated by the biomedical potential of these widespread intracellular bacteria, which are now used to control vector-borne diseases, while their symbiotic interaction with human parasites is targeted to fight filariasis, one of the most debilitating neglected tropical diseases ( 2 ). Horizontal transfers across species boundaries and the ability of these bacteria to be vertically transmitted through the egg are key elements of their successful pandemic. Involved in different types of symbiotic interactions, from parasitic to mutualistic, they influence the germ line biology of their host and the reproductive outcome through elaborate manipulations and integrate embryonic fate maps to navigate towards specific somatic targets and germ line precursors. Stably maintained in host populations, Wolbachia organisms also affect their hosts’ evolution, physiology, immunity, and development ( 3 ). While most studies have focused on the phenotypes they induce, insights into their intracellular lifestyle are also emerging. This article provides an overview of the molecular data and the current understanding of the cell biology underlying the mechanisms used by Wolbachia endosymbionts in arthropod and nematode hosts to target and subvert the germ line and reproduction machinery in order to support their transmission. The article also reviews the basis of their intracellular lifestyle in parasitic and mutualistic symbiotic interactions.

Unlike viruses, which can fuse with the plasma membrane to enter host cells, bacteria have only one way to penetrate a cell while preserving their integrity: engulfment through the invagination and closure of the plasma membrane. Therefore, the very first seconds of bacterial intracellular life inevitably occur within a vacuole. From there, two paths open: either to stay inside this vacuole, separated from the host cytoplasm, or to breach the membranous barrier and multiply in the cytosol. Both strategies have been adopted through evolution, and some microbes can even multiply in both environments. Remaining in a vacuole protects the bacteria from some aspects of the cytosolic innate host defense and allows them to build an environment perfectly adapted to their needs. However, this comes at a high price: the host resources are not readily accessible, as they cannot permeate the lipid bilayer that surrounds the bacteria. In addition, the area of this lipid bilayer needs to expand to accommodate bacterial multiplication. This requires building material and energy that are not directly invested in bacterial growth.

Infectious diseases are the second most important cause of death worldwide ( 1 ). Public health measures are partially successful in managing infectious disease burden, but major obstacles remain. Efficacious vaccines for major pathogens are still lacking ( 2 – 4 ), and the dramatic decline in the development of novel antimicrobials over the last 20 years ( 5 ) together with rapidly rising antimicrobial resistance is substantially reducing treatment options ( 6 ). The emerging crisis in infectious diseases is a major threat to human health.

Phagocytosis is an effective countermeasure exerted primarily by host macrophages and neutrophils to internalize and degrade microbes. Following uptake, most microbes are contained in a membrane-bound compartment called a phagosome, and through a series of tightly orchestrated events, nascent phagosomes mature and eventually fuse with lysosomes. Intracellular pathogens are unique in that they have strategies to avoid lysosomal degradation. Coevolution at the host-pathogen interface has enriched for mechanisms that allow intracellular pathogens to manipulate phagosome maturation and convert the host-derived compartment into a specialized organelle that supports replication. This specialized organelle is referred to as a pathogen-containing vacuole (PCV). Conversion of the nascent phagosome into a PCV is largely achieved by pathogen-directed subversion of host membrane transport.

Many bacterial pathogens have evolved to infect host cells from the inside. In fact, some bacteria, such as Rickettsia spp. and Coxiella spp., are entirely reliant on host intracellular resources to propagate ( 1 ). The adaptation of bacteria to an intracellular lifecycle is thought to confer a means to avoid the harsh extracellular milieu (low pH, physical stress, host defenses), to gain access to a nutrient-rich environment, and to facilitate the spread of the pathogen to neighboring host tissues ( 2 , 3 ).

Chromatin is located within the nuclei of eukaryotic cells and is composed of DNA wrapped around histone proteins. The highly ordered compaction of chromatin is crucial for the different functions encoded by the genetic material. These range from maintaining cell identity and genome integrity to adapting to environmental stimuli and cell replication. At the center of the chromatin language is its structural organization. This depends on the position and reversible covalent modifications to histone proteins and their cross talk with DNA and regulatory proteins. The basic unit of chromatin is the nucleosome, which is composed of an octamer of four histone proteins (H2A, H2B, H3, and H4) around which ∼147 bases of DNA are wrapped, with the linker histone (H1) outside the core structure providing structural integrity to the complex. Nucleosome remodelers are ATP-dependent enzymes that modify the chromatin structure through translocation, eviction, and introduction of histone variants ( 1 , 2 ), while histone-modifying enzymes introduce reversible covalent posttranslational modifications (PTMs) to histone tails.

The nervous system comprises the central nervous system (CNS) and the peripheral nervous system (PNS). Although many bacterial pathogens are known to invade the CNS and cause associated neuropathologies, much less is known about their intracellular manipulation of neural cells, particularly early events of bacterial infections, and how such bacterium-induced neural cell alterations could lead to bacterial survival, persistence, and the progression of infection as well as pathogenesis. A majority of the studies with these bacterial pathogens are immune-centric and focused on inflammatory aspects of nervous system diseases, and many reviews are available elsewhere with more detail on inflammatory and immune mechanisms of this bacteria-induced neurodegeneration ( 1 – 3 ).

MicroRNAs (miRNAs) are a class of small noncoding RNAs (typically 20 to 22 nucleotides long) that posttranscriptionally regulate the expression of target mRNAs exhibiting partially complementary binding sites ( 1 ). miRNAs are found in a wide range of organisms, including animals, plants, and viruses. According to the latest release of miRBase (http://www.mirbase.org/; release 22 March 2018), a total of 48,885 mature miRNAs are currently annotated in 271 species; 2,694 mature miRNAs are annotated in the human genome.

The human obligate intracellular pathogen Chlamydia trachomatis is the most frequent cause of sexually transmitted bacterial infection, with over 130 million cases per year ( 1 ). C. trachomatis causes blinding trachoma and pelvic inflammatory disease, the latter being causally connected with infertility and ectopic pregnancy ( 2 , 3 ). Pelvic inflammatory disease has further been linked to the occurrence of ovarian cancer ( 4 ); likewise, direct associations of C. trachomatis to ovarian and cervical carcinoma have been reported ( 5 – 7 ).

Outside a mammalian host, bacteria face numerous challenges that can result in life-threatening risks. These challenges include variations in temperature and osmolarity, predation, desiccation, and nutrient shortage. For bacteria with the ability to survive within a mammalian host, several of these threats are less severe, as host cells exist at a fixed temperature, osmolarity, and water and nutrient content. However, there is a cost associated with the benefits of an intracellular lifestyle. That cost is the threat of host-encoded immune defenses. Here, we describe the molecular mechanisms used by mammalian hosts to detect bacterial infection. We discuss the receptors encoded by the host immune system that recognize infection and the bacterial molecules that these receptors detect. Finally, we illustrate how these detection strategies, which have diverse mechanisms of action, share a thematically similar goal. This goal is to induce inflammatory responses that are typified by the recruitment of the biggest threat to bacterial viability to the sites of infection—polymorphonuclear leukocytes, also known as neutrophils.

The innate immune system comprises different mechanisms that recognize, restrict, and even kill intracellular bacteria. While most of these mechanisms aim at eradicating the infecting pathogen while maintaining cellular integrity, some also result in the concomitant death of the infected cell. The latter mechanism is exemplified by the assembly and activation of inflammasome complexes ( 1 ). The term “inflammasome” was coined in the early 2000s to describe multiprotein complexes that are assembled in the cytosol of activated macrophages and that serve as activation platforms for the cysteine protease caspase-1 ( 2 ). They are assembled by cytosolic sensor proteins that detect the presence of pathogen- or microbe-derived molecular patterns in the host cell cytosol, endogenous danger signals, or even disturbances of cellular homeostasis, so-called homeostasis-altering processes ( 1 , 3 ).

Immunocompetent mice harbor several layers of immune defense that can promote rejection of cell and tissue xenografts; these include innate mechanisms (mediated by complement, macrophage, and neutrophils) as well as adaptive immune responses (T cell-mediated and antibody-mediated rejection). In addition, resident tissues in the mouse harbor self-renewing stem cells and their differentiated progeny, which can effectively compete with human cells for endogenous tissue resources (physical space, nutrients, growth factors, etc.) that may play a role in sustaining human xenografts. As such, the development of “humanized” mouse models (human immune system [HIS] mice) is closely associated with the history of mutant mouse strains that harbor defects in hematopoietic system development and function. In recent decades, knowledge of the molecular mechanisms that regulate innate and adaptive immunity has led to the development of mouse models with an ever-increasing capacity to engraft human cells and tissues (reviewed in references 1 – 4 ). With respect to engraftment of the human hematopoietic system, as the severity of the immune deficiency in the mouse host has increased, the efficiency and durability of human hematopoietic cell “take” have improved remarkably, and importantly, a diverse compartment comprising many unique human immune subsets (including not only lymphocytes but also myeloid cells) has been achieved ( Fig. 1 ).

With each big scientific advancement, plaudits are quickly heaped on the scientists producing these great results as well as recognizing those that came before. Emerging technologies and technological advancements, often crucial to these ground-breaking discoveries, are often overlooked as merely being tools. Each big step forward in our understanding of host-pathogen interaction has been enabled in some way by advancements in technology. These advancements have allowed scientists to look at their problems in a new light, given us more detailed read-outs, or pushed us further toward the clinical environment. Microscopy is a clear example of these types of advancements. The development of the first primitive microscope in the 1670s allowed Antoine van Leeuwenhoek to describe the “animalcules” he saw, which were, in fact, bacteria being visualized for the first time using light-based microscopy ( 1 , 2 ).

Cellular imaging encompasses a large variety of methods that can be considered to be among the most powerful tools for investigating the molecular and cellular details of host-pathogen interactions, particularly when microbes display an intracellular lifestyle. This field has been expanding rapidly during the last 20 years and will likely gain further relevance as it continues to develop. Optical, fluorescence-based approaches are especially popular among infection biologists. In addition, we are witnessing a renaissance of electron microscopy (EM) due to numerous novel ultrastructural approaches changing the perception of EM from that of a confirmatory approach to that of a tool that drives new biological questions.

Investigations into the mechanisms that influence interactions between microbes and their hosts have exploded over the past ∼50 years. These approaches have led to a significantly greater understanding of how pathogens infect their hosts. Data from these experiments can influence therapeutic interventions and aid public health-mediated disease control on local and global scales. Progress in this field has been significant, driven by breakthroughs in several other areas, including molecular biology, biochemistry, immunology, cell biology, and genomics. Each of these independent areas has been underpinned by parallel breakthroughs in laboratory techniques alongside advances in the supporting computational biology (bioinformatics). Progress is ongoing in all of these fields and is continuing apace. Such progress is particularly important as a response to new infection-related threats, such as antimicrobial resistance and the emergence and/or discovery of new pathogens, which challenge our ability to maintain the disease control status quo ( 1 – 3 ).