Mbio
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Transformation of genetic material between bacteria was first observed in the 1920s using Streptococcus pneumoniae as a model organism. Since then, the mechanism of competence induction and transformation has been well characterized, mainly using planktonic bacteria or septic infection models. However, epidemiological evidence suggests that genetic exchange occurs primarily during pneumococcal nasopharyngeal carriage, which we have recently shown is associated with biofilm growth, and is associated with cocolonization with multiple strains. However, no studies to date have comprehensively investigated genetic exchange during cocolonization in vitro and in vivo or the role of the nasopharyngeal environment in these processes. In this study, we show that genetic exchange during dual-strain carriage in vivo is extremely efficient (10(-2)) and approximately 10,000,000-fold higher than that measured during septic infection (10(-9)). This high transformation efficiency was associated with environmental conditions exclusive to the nasopharynx, including the lower temperature of the nasopharynx (32 to 34°C), limited nutrient availability, and interactions with epithelial cells, which were modeled in a novel biofilm model in vitro that showed similarly high transformation efficiencies. The nasopharyngeal environmental factors, combined, were critical for biofilm formation and induced constitutive upregulation of competence genes and downregulation of capsule that promoted transformation. In addition, we show that dual-strain carriage in vivo and biofilms formed in vitro can be transformed during colonization to increase their pneumococcal fitness and also, importantly, that bacteria with lower colonization ability can be protected by strains with higher colonization efficiency, a process unrelated to genetic exchange. ⋯ Although genetic exchange between pneumococcal strains is known to occur primarily during colonization of the nasopharynx and colonization is associated with biofilm growth, this is the first study to comprehensively investigate transformation in this environment and to analyze the role of environmental and bacterial factors in this process. We show that transformation efficiency during cocolonization by multiple strains is very high (around 10(-2)). Furthermore, we provide novel evidence that specific aspects of the nasopharyngeal environment, including lower temperature, limited nutrient availability, and epithelial cell interaction, are critical for optimal biofilm formation and transformation efficiency and result in bacterial protein expression changes that promote transformation and fitness of colonization-deficient strains. The results suggest that cocolonization in biofilm communities may have important clinical consequences by facilitating the spread of antibiotic resistance and enabling serotype switching and vaccine escape as well as protecting and retaining poorly colonizing strains in the pneumococcal strain pool.
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New treatments are needed for extensively drug-resistant (XDR) Gram-negative bacilli (GNB), such as Acinetobacter baumannii. Toll-like receptor 4 (TLR4) was previously reported to enhance bacterial clearance of GNB, including A. baumannii. However, here we have shown that 100% of wild-type mice versus 0% of TLR4-deficient mice died of septic shock due to A. baumannii infection, despite having similar tissue bacterial burdens. The strain lipopolysaccharide (LPS) content and TLR4 activation by extracted LPS did not correlate with in vivo virulence, nor did colistin resistance due to LPS phosphoethanolamine modification. However, more-virulent strains shed more LPS during growth than less-virulent strains, resulting in enhanced TLR4 activation. Due to the role of LPS in A. baumannii virulence, an LpxC inhibitor (which affects lipid A biosynthesis) antibiotic was tested. The LpxC inhibitor did not inhibit growth of the bacterium (MIC>512 µg/ml) but suppressed A. baumannii LPS-mediated activation of TLR4. Treatment of infected mice with the LpxC inhibitor enhanced clearance of the bacteria by enhancing opsonophagocytic killing, reduced serum LPS concentrations and inflammation, and completely protected the mice from lethal infection. These results identify a previously unappreciated potential for the new class of LpxC inhibitor antibiotics to treat XDR A. baumannii infections. Furthermore, they have far-reaching implications for pathogenesis and treatment of infections caused by GNB and for the discovery of novel antibiotics not detected by standard in vitro screens. ⋯ Novel treatments are needed for infections caused by Acinetobacter baumannii, a Gram-negative bacterium that is extremely antibiotic resistant. The current study was undertaken to understand the immunopathogenesis of these infections, as a basis for defining novel treatments. The primary strain characteristic that differentiated virulent from less-virulent strains was shedding of Gram-negative lipopolysaccharide (LPS) during growth. A novel class of antibiotics, called LpxC inhibitors, block LPS synthesis, but these drugs do not demonstrate the ability to kill A. baumannii in vitro. We found that an LpxC inhibitor blocked the ability of bacteria to activate the sepsis cascade, enhanced opsonophagocytic killing of the bacteria, and protected mice from lethal infection. Thus, an entire new class of antibiotics which is already in development has heretofore-unrecognized potential to treat A. baumannii infections. Furthermore, standard antibiotic screens based on in vitro killing failed to detect this treatment potential of LpxC inhibitors for A. baumannii infections.
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Respiratory syncytial virus (RSV) is a leading cause of infant mortality worldwide. Toll-like receptor 4 (TLR4), a signaling receptor for structurally diverse microbe-associated molecular patterns, is activated by the RSV fusion (F) protein and by bacterial lipopolysaccharide (LPS) in a CD14-dependent manner. TLR4 signaling by LPS also requires the presence of an additional protein, MD-2. Thus, it is possible that F protein-mediated TLR4 activation relies on MD-2 as well, although this hypothesis has not been formally tested. LPS-free RSV F protein was found to activate NF-κB in HEK293T transfectants that express wild-type (WT) TLR4 and CD14, but only when MD-2 was coexpressed. These findings were confirmed by measuring F-protein-induced interleukin 1β (IL-1β) mRNA in WT versus MD-2(-/-) macrophages, where MD-2(-/-) macrophages failed to show IL-1β expression upon F-protein treatment, in contrast to the WT. Both Rhodobacter sphaeroides LPS and synthetic E5564 (eritoran), LPS antagonists that inhibit TLR4 signaling by binding a hydrophobic pocket in MD-2, significantly reduced RSV F-protein-mediated TLR4 activity in HEK293T-TLR4-CD14-MD-2 transfectants in a dose-dependent manner, while TLR4-independent NF-κB activation by tumor necrosis factor alpha (TNF-α) was unaffected. In vitro coimmunoprecipitation studies confirmed a physical interaction between native RSV F protein and MD-2. Further, we demonstrated that the N-terminal domain of the F1 segment of RSV F protein interacts with MD-2. These data provide new insights into the importance of MD-2 in RSV F-protein-mediated TLR4 activation. Thus, targeting the interaction between MD-2 and RSV F protein may potentially lead to novel therapeutic approaches to help control RSV-induced inflammation and pathology. ⋯ This study shows for the first time that the fusion (F) protein of respiratory syncytial virus (RSV), a major cause of bronchiolitis and death, particularly in infants and young children, physically interacts with the Toll-like receptor 4 (TLR4) coreceptor, MD-2, through its N-terminal domain. We show that F protein-induced TLR4 activation can be blocked by lipid A analog antagonists. This observation provides a strong experimental rationale for testing such antagonists in animal models of RSV infection for potential use in people.
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Pathogenic bacteria require iron for replication within their host. Klebsiella pneumoniae and other Gram-negative pathogens produce the prototypical siderophore enterobactin (Ent) to scavenge iron in vivo. In response, mucosal surfaces secrete lipocalin 2 (Lcn2), an innate immune protein that binds Ent to disrupt bacterial iron acquisition and promote acute inflammation during colonization. A subset of K. pneumoniae isolates attempt to evade Lcn2 by producing glycosylated Ent (Gly-Ent, salmochelin) or the alternative siderophore yersiniabactin (Ybt). However, these siderophores are not functionally equivalent and differ in their abilities to promote growth in the upper respiratory tract, lungs, and serum. To understand how Lcn2 exploits functional differences between siderophores, isogenic mutants of an Ent(+) Gly-Ent(+) Ybt(+) K. pneumoniae strain were inoculated into Lcn2(+/+) and Lcn2(-/-) mice, and the pattern of pneumonia was examined. Lcn2 effectively protected against the iroA ybtS mutant (Ent(+) Gly-Ent(-) Ybt(-)). Lcn2(+/+) mice had small foci of pneumonia, whereas Lcn2(-/-) mice had many bacteria in the perivascular space. The entB mutant (Ent(-) Ybt(+) Gly-Ent(-)) caused moderate bronchopneumonia but did not invade the transferrin-containing perivascular space. Accordingly, transferrin blocked Ybt-dependent growth in vitro. The wild type and the iroA mutant, which both produce Ent and Ybt, had a mixed phenotype, causing a moderate bronchopneumonia in Lcn2(+/+) mice and perivascular overgrowth in Lcn2(-/-) mice. Together, these data indicate that Lcn2, in combination with transferrin, confines K. pneumoniae to the airways and prevents invasion into tissue containing the pulmonary vasculature. ⋯ Gram-negative bacteria are a common cause of severe hospital-acquired infections. To cause disease, they must obtain iron and secrete the small molecule enterobactin to do so. Animal models of pneumonia using Klebsiella pneumoniae indicate that enterobactin promotes severe disease. Accordingly, the host defense protein lipocalin 2 exploits this common target by binding enterobactin and disrupting its function. However, pathogenic bacteria often make additional siderophores that lipocalin 2 cannot bind, such as yersiniabactin, which could make this host defense ineffective. This work compares the pattern and severity of pneumonia caused by K. pneumoniae based on which siderophores it produces. The results indicate that enterobactin promotes growth around blood vessels that are rich in the iron-binding protein transferrin, but yersiniabactin does not. Together, transferrin and lipocalin 2 protect this space against all types of K. pneumoniae tested. Therefore, the ability to acquire iron determines where bacteria can grow in the lung.
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The voluntary moratorium on gain-of-function research related to the transmissibility of highly pathogenic H5N1 influenza virus should continue, pending the resolution of critical policy questions concerning the rationale for performing such experiments and how best to report their results. The potential benefits and risks of these experiments must be discussed and understood by multiple stakeholders, including the general public, and all decisions regarding such research must be made in a transparent manner.