The goat livestock production chain involves more than one million rural establishments in the Brazilian Northeast, having a relevant socioeconomic function, whether directly to feed families or through the generation of income. Economic activity of strategic importance in this region has been directed to the production of leather, meat, milk, and their derivatives [1] [2] . Due to this upward economic movement, the occurrence of diseases in these animals deserves further studies targeted at enhancing productivity and profitability through effective disease control and prevention [3] .

Despite all research on the pathogenesis of caseous lymphadenitis caused by C. pseudotuberculosis, there is still inadequate information on the molecular mechanisms of bacterial pathogenesis and immunological prophylaxis against the infection. Identifying novel antigens may facilitate vaccine development and the formulation of innovative diagnostic tools for the detection of C. pseudotuberculosis in ovine and caprine samples [4] .

The discovery of the antigenic properties of proteins can help in understanding the pathogenicity, as well as factors associated with C. pseudotuberculosis resistance to antimicrobials. Studies on Heat Shock Proteins (HSPs), or chaperones, have demonstrated the important role of these molecules in increasing microbial resistance under stress conditions during infection. HSPs are considered immunologically essential because they are recognized by the host and are therefore capable of inducing strong cellular and humoral immune responses in mammals [5] . Studies have shown that these HSPs can be used as effective strategies to induce an immune response and as antigenic molecules for the development of vaccines against diseases [6] - [8] .

At the intracellular phase in the host, C. pseudotuberculosis faces several types of stresses inside the phagolysosome, characterized by the presence of low pH, high oxidative potential, thermal shock, nitrosative, osmotic, and other conditions. However, resistance to different environmental stresses is associated with the ability of C. pseudotuberculosis to survive in the host [7] [9] .

Therefore, the bacteria adapt to different stress conditions imposed by the host during infection. This review examines the expression of potential transcriptional genes in C. pseudotuberculosis that may shed light on the mechanisms underlying its virulence, resistance, and pathogenicity.

In the current review, findings were extracted from PubMed, NCBI, Scopus, Web of Science, and Google Scholar databases from early January 2012 to late May 2023 ( Figure 1 ), searching for keywords including “caseous lymphadenitis”, “stress”, “genes”, “C. pseudotuberculosis”, “resistance” and HSPs. These articles were subsequently filtered to ensure relevance to the scope of this review.

The articles were initially selected based on their title and abstract. The inclusion and exclusion criteria were then applied, and finally, the selected articles were read in full, as shown in Figure 1 . The inclusion criteria were full texts, studies written in English, and addressing Corynebacterium pseudotuberculosis, tolerance, adaptability, and resistance factors. Studies unrelated to caseous lymphadenitis were excluded. After reading the selected articles, relevant information that met the objective of the literature review was extracted.

Corynebacterium pseudotuberculosis is a Gram-positive bacterium that is part of a general group of the Actinobacteria class that includes the genus Mycobacterium, Nocardia, and Rhodococcus. It is the etiologic agent responsible for cases of Caseous Lymphadenitis (CL) affecting small ruminants (sheep and goats) all over the world. C. pseudotuberculosis is an intracellular facultative anaerobic pathogen that exhibits pleomorphic forms, such as coccoid and filamentous rods, characterized as a Gram-positive coccobacillus. These microorganisms are characterized by a layer of mycolic acids, known as the mycomembrane, which overlies a complex cell wall with a high DNA content and confers significant resistance to environmental stress. In addition, cell membrane components are crucial for the interaction of these bacteria with host cells associated with the synthesis and interaction of mycolic acid as components of the immune system of the genus Corynebacterium [10] [11] .

As biochemical characteristics, catalase and urease ferment glucose and ribose without gas production, and about 80% of the isolates ferment maltose, but not lactose, mannose, or sucrose. They do not produce spores and are immobile, possess pili, and are capable of producing biofilm [12] .

Strategically, this microorganism has a cell wall organization, characterized mainly by the presence of an immense complex polymer composed of peptidoglycan, arabinogalactan, and mycolic acids. This is related to the potential to survive for several weeks in the environment, which contributes to the ability to spread within a herd. The genomes of several species of this group have already been completely sequenced, especially due to the considerable medical, veterinary, and biotechnological importance of these organisms. Transmission between sheep or goats occurs mainly through contamination of superficial wounds, which can appear during common procedures, such as shearing or castration, or through injuries to the animal’s body caused by other traumatic events [13] [14] .

Gene processes and molecules are involved in resistance to stressful conditions. These include stress proteins and transcription factors, efflux pumps, altered membrane composition, energy metabolism, chemical detoxification, and chaperone synthesis and accumulation. Microbial organization and modulation determine a successful and tolerated response to stress, involving one or a few genes, with new genomic and biological tools aiding the understanding of resistance mechanisms [23] .

In C.pseudotuberculosis, the modulation of gene expression at the transcriptional level is carried out by a class of proteins that constitute dissociable subunits of RNA polymerase, the sigma (σ) factors. The alternative σ factors of bacterial RNA polymerase provide a means of rapidly regulating gene expression in response to various changes in the extracellular environment [26] .

Potential pathogenicity islands of C. pseudotuberculosis, a group of virulence genes, are essential for distinguishing pathogenic from nonpathogenic species. They are present in large numbers and contain species-identifying virulence elements. The formation of the virulence factors they contain may help increase the adaptability of strains to different host environments. This increased adaptability is demonstrated by the discovery of genes expressing proteins for iron uptake, production of fimbrial subunits, insertion elements, and secreted toxins, acquired through horizontal gene transfer, contributing to our understanding of how this species can resist environmental adversities [18] [27] .

Genetic composition of pathogenicity islands can shed light on the lifestyle of this bacterium, since they include virulence genes that mediate mechanisms of adhesion, invasion, colonization, proliferation in the host, and adaptation to the immune system. Furthermore, pathogenicity islands are characterized as unstable regions that can be affected by insertions and deletions, influencing bacterial adaptability to new environments and hosts. In C. pseudotuberculosis, σ factors are the main regulators of gene expression at the transcriptional level and, therefore, play an important role in the bacterial adaptive response to different environmental stimuli. These factors form a holoenzyme with the core RNA polymerase and direct it to specific promoters, resulting in the activation of selected genes. Most bacteria possess several different σ factors that allow them to maintain basal gene expression as well as modulation in response to specific environmental signals [18] .

The PhoPR (transmembrane sensory protein histidine kinase) system regulates the expression of several genes involved in metabolic, virulence, and resistance processes in several intracellular bacterial pathogens. These are regulatory proteins that function in bacteria as sensory and adaptive factors in response to a wide range of environmental stimuli. However, two-component systems, such as PhoP/PhoQ (intracellular response regulatory protein), control the transcription of key virulence genes essential for survival in host cells in several intracellular bacterial pathogens, including C. pseudotuberculosis sp., Salmonella sp., Shigella sp., Yersinia sp., and Mycobacterium tuberculosis. This makes it particularly remarkable that many microorganisms can tolerate significant fluctuations in the conditions necessary for their survival and growth [10] [27] .

Environmental change often requires the expression of new genes, activated by exposure to these conditions, a phenomenon called the stress response. These findings have significant applications across diverse areas, including infection biology, food microbiology, and biotechnology. Understanding how microorganisms respond to stresses caused by the immune system, antibiotics, antifungals, and disinfectants could lead to a better understanding of infectious diseases, including the identification of targets for future drug and vaccine development [28] .

HSPs (Heat Shock Proteins), also known as chaperones, are highly conserved proteins. Prokaryotic and eukaryotic cells synthesize heat shock proteins constitutively as a result of stressful conditions, including adverse environmental circumstances (radiation, acidity, osmotic changes, temperature changes). Studies show that HSPs have the capacity for intracellular interaction with many proteins, playing a role in folding and translocation, repair, and formation of complexes [29] .

Especially in bacteria, surface expression of HSPs and proteases is presumably essential to overcome changes involving protein denaturation. They are controlled by a specific sigma factor (rpoH gene), which provides a means of rapidly regulating HSP gene expression in response to various changes in the bacterial extracellular environment [30] [31] .

Bacterial HSPs are both protective and exert pathogenic activities in mammals depending on the infection. The most studied bacterial HSPs are HtpG, GroEL and HSP60, HSP70 (DnaK), HSP25, HSP90, Hsp65, Hsp10, HspR, Cpl-B and DnaJ. They exhibit varying molecular weights and have been shown to be highly immunogenic, capable of inducing antibody production, inflammatory responses, and activation of macrophages and T cells [7] [8] .

GroEL chaperone (HSP60) is essential for preserving cell growth under many adverse environmental conditions. It assists in several cellular processes, including DNA replication, mutagenesis on UV exposure, RNA transcription, and translocating proteins across membrane barriers and secretion. Studies with HSP60 analyzed the synthesis of specific serum antibodies in the host, demonstrating that bacterial HSP can be a biomarker to monitor immune status. Thus, the dual role of HSP60 as an immune modulator and a biomarker provides an opportunity to modulate immunity for therapeutic purposes and monitor the immune response in health and disease [32] [33] .

Assessing differential gene expressions of HSPs is useful for understanding the mechanisms of adaptation during host infection. Different transcripts may be expressed among strains C. pseudotuberculosis with diverse characteristics during infection. Certainly, it plays an important role in the adaptations and modifications required for the development of a successful infection by this pathogen. Factors that regulate these genes respond to specific environmental or cellular signals to stimulate or inhibit transcription or translation, modifying the rate of synthesis of gene products, which is important for the physiological and biochemical adaptation required [20] .

The immune response established for this bacterium is complex and involves both innate and adaptive immune response mechanisms. Macrophages and neutrophils stand out at the beginning of the infection and establish a protective response to primary and secondary infection. During the infection process, C. Pseudotuberculosis alters its gene expression to resist different types of stress and avoid the host’s immune system. The bacteria are phagocytosed by macrophages, which are drained into local lymph nodes. Experiments with lymph node drainage have shown that a large layer of macrophages and lymphocytes is formed around the necrotic tissue. Once internalized, bacteria avoid the cellular immune response mechanisms and can survive and multiply rapidly inside the macrophage phagosome [34] .

To comprehend the functional role of macrophage response to infection by C. pseudotuberculosis, it is important to emphasize that in the course of maturation, phagosomes acquire an arsenal of antimicrobial characteristics. In phagosome acidification, there is an increase in the synthesis of ATPases that acidify the phagosome lumen, creating a hostile environment that prevents microbial growth, not only directly impairing bacterial metabolism but also favoring the H+ gradient generated by this ATPase. This gradient is used to destroy essential nutrients present in the phagosomal lumen and can be used by this microorganism [30] . ATPase also facilitates the generation of superoxide (O₂), generating reactive oxygen species. However, bacterial defense mechanisms include modification of their surface to resist or break down antimicrobial peptides, and expression of enzymes such as catalase. This enzyme converts reactive species into less harmful compounds or prevents recruitment of protein complexes that synthesize Reactive Nitrogen Species (RNS) or Reactive Oxygen Species (ROS) [35] .

Factors that contribute to the success of the bacterium are the ability to resist potentially bactericidal host defenses and elimination by an activated immune system. This host-imposed resistance to abiotic stresses is in part due to the low permeability of the bacterial cell envelope to many toxic molecules. Furthermore, it depends on detoxifying reactive oxygen and nitrogen molecules produced by the host, repairing the damage these molecules cause, and maintaining a neutral intrabacterial pH in acidic environments [36] [37] .

Research shows that C. pseudotuberculosis can survive inside macrophages and resist aggressive host conditions, establishing infection and inducing typical clinical symptoms. One of the characteristics of resistance is the lipid cover recognized as an electron-dense layer that is found on the outside of the cell wall which plays an important role, suggesting that it provides a protective barrier against the antibacterial mechanisms of the host. This morphology helps to establish a regulatory mechanism that allows adaptation to environmental stimuli [43] .

By surviving inside macrophages for more than 48 hours, the bacteria are released as a result of a process that leads to death by phagocytes, although this property varies between different strains. These effects may be associated with the outer lipid layer in the C. pseudotuberculosis cell wall and other antigenic components that attenuate the production of nitric oxide by macrophages. However, bacterial growth becomes uncontrolled within the host cell, which tries to restrict and limit the infection through the formation of pyogranulomas, which are characterized by the encapsulation of cells infected by C. pseudotuberculosis [44] [45] .

Changes in environmental conditions affect the pathogenic properties of microorganisms that lead to the synthesis of HSPs (heat shock), which biochemically change their molecular structure [45] . Especially in bacteria, the expression of HSPs and proteases is essential to overcome the changes that involve the denaturation of proteins. They are controlled by a specific sigma factor, which provides a means of rapidly regulating the gene expression of HSPs in response to various changes in the bacterial extracellular environment. However, it is still not clear what these proteins would be in C. pseudotuberculosis [34] [46] .

Functional genomics approaches focused on pathogen interactions with macrophages further enhance our understanding of pathogenicity. Corynebacterium pseudotuberculosis is able to survive and grow in animal macrophages in order to spread within the host, similarly to C. diphtheriae, which is able to survive inside human macrophages, ensuring growth and survival [47] . Since proteins exposed and secreted on the cell surface play an essential role in the host-pathogen interaction, these proteins represent a potential target in the formulation of drugs, biomarkers, and vaccines [48] [49] .

An immune response was observed after administration of the C. Pseudotu-berculosis DNA-hsp60 vaccine in mice, utilizing genes encoding HSPs to induce immunity. The results indicated that the DNA-hsp60 vaccine generated IgG1 and IgG2a responses when administered to BALB/c mice, but with a tendency toward a Th1-type immune response after 30 days of the first immunization. The protective efficacy of DNA-hsp65 vaccines has also been extensively studied for Mycobacterium tuberculosis. Another example was the M. leprae DNA-hsp65 vaccine, which induced protective immunity against tuberculosis challenge in a murine model. Similarly, the M. avium DNA-hsp65 vaccine induced a strong protective immune response in lambs and protected against infection with tuberculosis of the M. avium subspecies [11] [19] [50] .

C. pseudotuberculosis strains can alter gene expression as a form of adaptation under stress conditions. These genes possibly encode proteins that are related to the survival of the bacteria under adverse conditions [35] [51] . The expression of potential genes (Hsp65, Hsp10, HspR, Dna-J, Cpl-B) may support the tolerance of C. pseudotuberculosis to the hostile conditions encountered during infection. These genes may be related to the regulation of metabolic functions, growth, virulence, and immunomodulation. Thus, they may contribute to the understanding of new immunization strategies and drugs for controlling caseous lymphadenitis [8] .

The use of the molecular biology tool can help with the identification of these genes associated with the propagation and persistence of C. pseudotuberculosis during the infection process. It may also suggest possible paths for studies related to the development of vaccines, diagnoses, and therapies that can help to minimize the damage of the disease.